Immune cells engineered to express cd19-directed chimeric antigen receptors and uses thereof in immunotherapy

ABSTRACT

Provided for herein in several embodiments are immune cell-based (e.g., natural killer (NK) cell) compositions comprising CD19-directed chimeric antigen receptors. In some, embodiments the anti-CD19 binder portion of the CAR is humanized. In several embodiments, the humanized anti-CD19 CAR expressing cells exhibit enhanced expression of the CAR as well as enhanced cytotoxicity and/or persistence. Several embodiments include methods of using of the anti-CD19 CAR expressing immune cells in immunotherapy.

RELATED CASES

This application is a continuation of International Patent ApplicationNo. PCT/US2020/020824, filed Mar. 3, 2020, which claims the priority toU.S. Provisional Patent Application Nos. 62/814,180, filed Mar. 5, 2019,62/895,910, filed Sep. 4, 2019, and 62/932,165, filed Nov. 7, 2019, theentire contents of each of which is incorporated by reference herein.

FIELD

Some embodiments of the methods and compositions provided herein relateto CD19-directed receptors. In some, embodiments the receptors arechimeric. Some embodiments include methods of use of the chimericreceptors in immunotherapy.

BACKGROUND

As further knowledge is gained about various cancers and whatcharacteristics a cancerous cell has that can be used to specificallydistinguish that cell from a healthy cell, therapeutics are underdevelopment that leverage the distinct features of a cancerous cell.Immunotherapies that employ engineered immune cells are one approach totreating cancers.

INCORPORATION BY REFERENCE OF MATERIAL IN ASCII TEXT FILE

This application incorporates by reference the Sequence Listingcontained in the following ASCII text file being submitted concurrentlyherewith: File Name: NKT033C2_ST25.txt; created Nov. 3, 2020, 435 KB insize.

SUMMARY

Immunotherapy presents a new technological advancement in the treatmentof disease, wherein immune cells are engineered to express certaintargeting and/or effector molecules that specifically identify and reactto diseased or damaged cells. This represents a promising advance due,at least in part, to the potential for specifically targeting diseasedor damaged cells, as opposed to more traditional approaches, such aschemotherapy, where all cells are impacted, and the desired outcome isthat sufficient healthy cells survive to allow the patient to live. Oneimmunotherapy approach is the recombinant expression of chimericreceptors in immune cells to achieve the targeted recognition anddestruction of aberrant cells of interest.

In several embodiments, there is provided herein an immune cell, andalso populations of immune cells, that expresses a CD19-directedchimeric receptor, the chimeric receptor comprising an extracellularanti-CD19 binding moiety, a hinge and/or transmembrane domain, and anintracellular signaling domain. Also provided for herein arepolynucleotides (as well as vectors for transfecting cells with thesame) encoding a CD19-directed chimeric antigen receptor, the chimericantigen receptor comprising an extracellular anti-CD19 binding moiety, ahinge and/or transmembrane domain, and an intracellular signalingdomain.

In several embodiments, there is provided a polynucleotide encoding aCD19-directed chimeric antigen receptor, the chimeric antigen receptorcomprising an extracellular anti-CD19 binding moiety, wherein theanti-CD19 binding moiety comprises a variable heavy (VH) domain of asingle chain Fragment variable (scFv) and a variable light (VL) domainof a scFv, a hinge, a transmembrane domain, and an intracellularsignaling domain, and wherein the intracellular signaling domaincomprises an OX40 subdomain, a CD3 zeta subdomain.

In several embodiments, there is provided a polynucleotide encoding aCD19-directed chimeric antigen receptor, the chimeric antigen receptorcomprising an extracellular anti-CD19 binding moiety, wherein theanti-CD19 binding moiety comprises a variable heavy (VH) domain of asingle chain Fragment variable (scFv) and a variable light (VL) domainof a scFv, wherein the encoded VH domain comprises at least one heavychain complementarity determining region (CDR) selected from the groupconsisting of SEQ ID NO: 133, SEQ ID NO: 134, and SEQ ID NO: 135,wherein the encoded VL domain comprises at least one light chaincomplementarity determining region (CDR) selected from the groupconsisting of SEQ ID NO: 127, SEQ ID NO: 128, and SEQ ID NO: 129, ahinge domain, a transmembrane domain, an intracellular signaling domain,and wherein the intracellular signaling domain comprises an OX40subdomain and a CD3 zeta subdomain.

In several embodiments, the polynucleotide also encodes membrane-boundinterleukin-15 (mbIL15). However, in some embodiments, a separatepolynucleotide is used to encode the mbIL15. In several embodiments, thetransmembrane domain is derived from or comprises a CD8 alphatransmembrane domain. In several embodiments, the CD8 alphatransmembrane domain is encoded by SEQ ID NO: 3. In several embodiments,the hinge is derived from or comprises a CD8 alpha hinge. In severalembodiments, the CD8 alpha hinge is encoded by SEQ ID NO: 1. In severalembodiments, the OX40 subdomain is encoded by a sequence having at least90% (e.g., 90-95%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQID NO. 5. In several embodiments, the CD3 zeta subdomain is encoded by asequence having at least 90% (e.g., 90-95%, 95%, 96%, 97%, 98%, or 99%)sequence identity to SEQ ID NO. 7. In several embodiments, the mbIL15 isencoded by a sequence having at least 90% (e.g., 90-95%, 95%, 96%, 97%,98%, or 99%) sequence identity to SEQ ID NO. 11. In several embodiments,the OX40 domain is encoded by SEQ ID NO: 5, the CD3 zeta subdomain isencoded by SEQ ID NO. 7 and/or mbIL15 (whether encoded separately orbicistronically) is encoded by SEQ ID NO: 11. In several embodiments,the encoded OX40 subdomain comprises the amino acid sequence of SEQ IDNO: 6, the encoded CD3 zeta subdomain comprises the amino acid sequenceof SEQ ID NO: 8, and/or the encoded mbIL15 (whether encoded separatelyor bicistronically) comprises the amino acid sequence of SEQ ID NO:

12.

In several embodiments, the VH domain comprises a VH domain selectedfrom SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, and SEQ ID NO: 123,and wherein the VL domain comprises a VL domain selected from SEQ ID NO:117, SEQ ID NO: 118, and SEQ ID NO: 119. In several embodiments, thepolynucleotide encodes a VL domain comprising at least one light chaincomplementarity determining region (CDR) selected from the groupconsisting of SEQ ID NO: 127, SEQ ID NO: 128, and SEQ ID NO: 129. Inseveral embodiments, the polynucleotide encodes a VH domain comprisingat least one heavy chain complementarity determining region (CDR)selected from the group consisting of SEQ ID NO: 133, SEQ ID NO: 134,and SEQ ID NO: 135. In several embodiments, the polynucleotide isdesigned (e.g., engineered) to reduce potential antigenicity of theencoded protein and/or enhance one or more characteristics of theencoded protein (e.g., target recognition and/or bindingcharacteristics) Thus, according to several embodiments, the anti-CD19binding moiety does not comprise certain sequences. For example,according to several embodiments the polynucleotide does not encode oneor more of SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40,SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO:45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ IDNO: 50, SEQ ID NO: 51, SEQ ID NO: 52. SEQ ID NO: 53, SEQ ID NO: 54, orSEQ ID NO: 55. In several embodiments, the encoded VH domain comprisesan amino acid sequence at having at least 90% (e.g., 90-95%, 95%, 96%,97%, 98%, or 99%) sequence identity to SEQ ID NO: 120. In severalembodiments, the encoded VH domain comprises the amino acid sequence ofSEQ ID NO: 120. In several embodiments, the encoded VL domain comprisesan amino acid sequence at having at least 90% (e.g., 90-95%, 95%, 96%,97%, 98%, or 99%) sequence identity to SEQ ID NO: 118. In severalembodiments, the encoded VL domain comprises the amino acid sequence ofSEQ ID NO: 118. In several embodiments, VH domain is derived from aparent amino acid sequence and is modified from the parent sequence. Forexample, mutations, truncations, extensions, conservative substitutions,or other modifications are introduced to increase the affinity of thedomain for its target, increase the avidity for the target, and/orreduce potential antigenicity of the sequence. In several embodiments,the VH domain results from humanization of the VH domain amino acidsequence set forth in SEQ ID NO: 33. Likewise, in several embodiments,the VL domain results from humanization of the VL domain amino acidsequence set forth in SEQ ID NO: 32. In several embodiments, thepolynucleotide encodes a CD19-directed chimeric antigen receptor havingat least is encoded by a sequence having at least 90% (e.g., 90-95%,95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequenceset forth in SEQ ID NO: 187. In several embodiments, the polynucleotideencodes a CD19-directed chimeric antigen receptor comprising the aminoacid sequence set forth in SEQ ID NO: 187.

In several embodiments, the polynucleotide does not encode or otherwisecomprise a DAP10 domain. In several embodiments, the polynucleotide doesnot encode or otherwise comprise a DAP12 domain. In several embodiments,the intracellular signaling domain comprises additional subdomains, thatadvantageously enhance generation of cytotoxic signals by cellsexpressing the constructs. In several embodiments, the polynucleotidefurther encodes one or more of CD44 and CD27 as signaling subdomains. Inseveral embodiments, the polynucleotide optionally further encodes adetection tag or other moiety (e.g., marker) that allows for detectionof expression of the protein(s) encoded by the polynucleotide by hostcells.

There are also provided for herein uses of the disclosed polynucleotidesin the manufacture of a medicament for enhancing NK cell cytotoxicity ina mammal in need thereof, in the manufacture of a medicament fortreating cancer in a mammal in need thereof and/or for the treatment ofcancer in a mammal in need thereof.

There are also provided for herein engineered immune cells that expressthe CD19-directed chimeric antigen receptors encoded by thepolynucleotides disclosed herein. In several embodiments, the engineeredimmune cells are natural killer (NK) cell. In some embodiments, theengineered cells are T cells, though combinations of NK cell and T cells(and optionally other immune cell types) are used in some embodiments.In several embodiments, the immune cells are allogeneic with respect toa subject receiving the cells. There are also provided for herein theuse of immune cells that express the CD19-directed chimeric antigenreceptors encoded by the polynucleotides disclosed herein for thetreatment of cancer in a mammal in need thereof. There are also providedfor herein the use of immune cells that express the CD19-directedchimeric antigen receptors encoded by the polynucleotides disclosedherein for the manufacture of a medicament for the treatment of cancerin a mammal in need thereof.

In several embodiments, there are provided methods for treating cancerusing the polynucleotides disclosed herein. For example, in severalembodiments the methods comprise administering to a subject having acancer a composition comprising a population of immune cells expressingCD19-directed chimeric antigen receptors as disclosed herein. In severalembodiments, the CAR comprises an extracellular anti-CD19 bindingmoiety, wherein the anti-CD19 binding moiety comprises a variable heavy(VH) domain of a single chain Fragment variable (scFv) and a variablelight (VL) domain of a scFv, a hinge, such as a CD8 alpha hinge, atransmembrane domain, such as a CD8 alpha transmembrane domain; and anintracellular signaling domain comprising an OX40 subdomain and a CD3zeta subdomain, and wherein the cell also expresses membrane-boundinterleukin-15 (mbIL15). In several embodiments, the OX40 subdomain isencoded by a sequence having at least 95% sequence identity to SEQ IDNO. 5, the CD3 zeta subdomain is encoded by a sequence having at least95% sequence identity to SEQ ID NO. 7, and/or the mbIL15 is encoded by asequence having at least 95% sequence identity to SEQ ID NO. 11. Inseveral embodiments, the encoded VH domain comprises an amino acidsequence having at least 95% sequence identity to SEQ ID NO: 120,wherein the encoded VL domain comprises an amino acid sequence having atleast 95% sequence identity to SEQ ID NO: 118. In several embodiments,the polynucleotide encodes a CD19-directed chimeric antigen receptorhaving at least 95% sequence identity to the amino acid sequence setforth in SEQ ID NO: 187. As discussed above, in several embodiments, theimmune cells expressing such CD19 CAR constructs are natural killer (NK)cells. In some embodiments, the immune cells are T cells. In severalembodiments, combinations of NK and T cells (and optionally other immunecells) are used. In several embodiments, the cells are allogeneic cellsoriginating from a donor that is not the subject. In severalembodiments, the cells are autologous cells originating the subject.Mixtures of allogeneic and autologous cells may also be used, in someembodiments. In several embodiments, the administered populationcomprises about 2×10⁶ cells per kilogram of body weight of the subject.In several embodiments, the administration is intravenous. In severalembodiments, the method further comprises administering one or moredoses of interleukin 2 to the subject. In several embodiments, themethods also involve the administration of another therapy to thesubject. For example, in several embodiments, the methods involveadministering a chemotherapy treatment to the subject prior to theadministration of the cells. In several embodiments, the chemotherapytreatment induces lymphodepletion in the subject. In some embodiments,the subject is administered a combination of cyclophosphamide andfludarabine prior to administration of the cells. In severalembodiments, the cyclophosphamide is administered in a dose betweenabout 400 and about 600 mg/m². In several embodiments, the fludarabineis administered in a dose between about 25 and 25 mg/m². In severalembodiments, the lymphodepleting chemotherapy is administered severaldays prior to administration of engineered immune cells and optionallyadministered multiple times. For example, in several embodiments thelymphodepleting chemotherapy is administered on at least the fifth,fourth, and/or third day prior to administration of engineered immunecells disclosed herein.

In several embodiments there is provided a polynucleotide encoding ahumanized CD19-directed chimeric antigen receptor, the chimeric antigenreceptor comprising a humanized anti-CD19 binding moiety, aco-stimulatory domain, and a signaling domain. In several embodiments,the co-stimulatory domain comprises OX40. In several embodiments, thehumanized anti-CD19-binding moiety comprises a humanized scFv, whereinone or more of the heavy and light chains have been humanized. Inseveral embodiments, one or more of the CDRs on the heavy and/or lightchains have been humanized. For example, in several embodiments, thereis provided a polynucleotide encoding a humanized CD19-directed chimericantigen receptor, the chimeric antigen receptor comprising anextracellular anti-CD19 binding moiety, wherein the anti-CD19 bindingmoiety comprises a heavy chain variable (VH) domain and a and a lightchain variable (VL) domain, the VH domain comprising a VH domainselected from SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, and SEQ IDNO: 123 and the VL domain comprising a VL domain selected from SEQ IDNO: 117, SEQ ID NO: 118, and SEQ ID NO: 119, a hinge and/ortransmembrane domain, an intracellular signaling domain.

In several embodiments, there is provided a polynucleotide encoding ahumanized chimeric antigen receptor (CAR), wherein the CAR comprises asingle chain antibody or single chain antibody fragment which comprisesa humanized anti-CD19 binding domain, a transmembrane domain, a primaryintracellular signaling domain comprising a native intracellularsignaling domain of CD3-zeta, or a functional fragment thereof, and acostimulatory domain comprising a native intracellular signaling domainof a protein selected from the group consisting of OX40, CD27, CD28,ICOS, and 4-1 BB, or a functional fragment thereof, wherein saidanti-CD19 binding domain comprises a light chain complementarydetermining region 1 (LC CDR1) of SEQ ID NO: 124, 127, or 130, a lightchain complementary determining region 2 (LC CDR2) of SEQ ID NO: 125,128, or 131, and a light chain complementary determining region 3 (LCCDR3) of SEQ ID NO: 126, 129, or 132, and a heavy chain complementarydetermining region 1 (HC CDR1) of SEQ ID NO: 133, 136, 139, or 142, aheavy chain complementary determining region 2 (HC CDR2) of SEQ ID NO:134, 137, 140, or 143, and a heavy chain complementary determiningregion 3 (HC CDR3) of SEQ ID NO: 135, 138, 141, or 144.

In several embodiments, there is provided a polynucleotide encoding ahumanized CD19-directed chimeric antigen receptor, the chimeric antigenreceptor comprising an extracellular anti-CD19 binding moiety, whereinthe anti-CD19 binding moiety comprises a humanized scFv sequencecomprising a variable light (VL) domain of SEQ ID NO: 117, a hingeand/or transmembrane domain, and an intracellular signaling domain. Inseveral embodiments, the polynucleotide encodes the humanized chimericantigen receptor of SEQ ID NO: 161, SEQ ID NO: 167, SEQ ID NO: 173, SEQID NO: 179, SEQ ID NO: 185, SEQ ID NO: 191, SEQ ID NO: 197, or SEQ IDNO: 203.

In several embodiments, there is provided a polynucleotide encoding ahumanized CD19-directed chimeric antigen receptor, the chimeric antigenreceptor comprising an extracellular anti-CD19 binding moiety, whereinthe anti-CD19 binding moiety comprises a humanized scFv sequencecomprising a variable light (VL) domain of SEQ ID NO: 118, a hingeand/or transmembrane domain, and an intracellular signaling domain. Inseveral embodiments, the polynucleotide encodes the humanized chimericantigen receptor of SEQ ID NO: 163, SEQ ID NO: 169, SEQ ID NO: 175, SEQID NO: 181, SEQ ID NO: 187, SEQ ID NO: 193, SEQ ID NO: 199, or SEQ IDNO: 205.

In several embodiments, there is provided a polynucleotide encoding ahumanized CD19-directed chimeric antigen receptor, the chimeric antigenreceptor comprising an extracellular anti-CD19 binding moiety, whereinthe anti-CD19 binding moiety comprises a humanized scFv sequencecomprising a variable light (VL) domain of SEQ ID NO: 119, a hingeand/or transmembrane domain, and an intracellular signaling domain. Inseveral embodiments, the polynucleotide encodes the humanized chimericantigen receptor of SEQ ID NO: 165, SEQ ID NO: 171, SEQ ID NO: 177, SEQID NO: 183, SEQ ID NO: 189, SEQ ID NO: 195, SEQ ID NO: 201, or SEQ IDNO: 207.

In several embodiments, there is provided a polynucleotide encoding ahumanized CD19-directed chimeric antigen receptor, the chimeric antigenreceptor comprising an extracellular anti-CD19 binding moiety, whereinthe anti-CD19 binding moiety comprises a humanized scFv sequencecomprising a variable heavy (VH) domain of SEQ ID NO: 120, a hingeand/or transmembrane domain, and an intracellular signaling domain. Inseveral embodiments, the polynucleotide encodes the humanized chimericantigen receptor of SEQ ID NO: 161, SEQ ID NO: 163, SEQ ID NO: 165, SEQID NO: 185, SEQ ID NO: 187, or SEQ ID NO: 189.

In several embodiments, there is provided a polynucleotide encoding ahumanized CD19-directed chimeric antigen receptor, the chimeric antigenreceptor comprising an extracellular anti-CD19 binding moiety, whereinthe anti-CD19 binding moiety comprises a humanized scFv sequencecomprising a variable heavy (VH) domain of SEQ ID NO: 121, a hingeand/or transmembrane domain, and an intracellular signaling domain. Inseveral embodiments, the polynucleotide encodes the humanized chimericantigen receptor of SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQID NO: 191, SEQ ID NO: 193, or SEQ ID NO: 195.

In several embodiments, there is provided a polynucleotide encoding ahumanized CD19-directed chimeric antigen receptor, the chimeric antigenreceptor comprising an extracellular anti-CD19 binding moiety, whereinthe anti-CD19 binding moiety comprises a humanized scFv sequencecomprising a variable heavy (VH) domain of SEQ ID NO: 122, a hingeand/or transmembrane domain, and an intracellular signaling domain. Inseveral embodiments, the polynucleotide encodes the humanized chimericantigen receptor of SEQ ID NO: 173, SEQ ID NO: 175, SEQ ID NO: 177, SEQID NO: 197, SEQ ID NO: 199, or SEQ ID NO: 201.

In several embodiments, there is provided a polynucleotide encoding ahumanized CD19-directed chimeric antigen receptor, the chimeric antigenreceptor comprising an extracellular anti-CD19 binding moiety, whereinthe anti-CD19 binding moiety comprises a humanized scFv sequencecomprising a variable heavy (VH) domain of SEQ ID NO: 123, a hingeand/or transmembrane domain, and an intracellular signaling domain. Inseveral embodiments, the polynucleotide encodes the humanized chimericantigen receptor of SEQ ID NO: 179, SEQ ID NO: 181, SEQ ID NO: 183, SEQID NO: 203, SEQ ID NO: 205, or SEQ ID NO: 207.

In several embodiments, the provided polynucleotides also encodemembrane-bound interleukin-15 (mbIL15).

In several embodiments, the intracellular signaling domain comprises anOX40 subdomain. However, in several embodiments the intracellularsignaling domain comprises one or more of an OX40 subdomain, a CD28subdomain, an iCOS subdomain, a CD28-41 BB subdomain, a CD27 subdomain,a CD44 subdomain, or combinations thereof.

In several embodiments, the chimeric antigen receptor comprises a hingeand a transmembrane domain, wherein the hinge is a CD8 alpha hinge,wherein the transmembrane domain is either a CD8 alpha or an NKG2Dtransmembrane domain. In several embodiments, the intracellularsignaling domain comprises a CD3zeta domain.

In several embodiments, the polynucleotide does not encode SEQ ID NO:112, 113, or 114. In several embodiments the polynucleotide does notencode SEQ ID NO: 116.

In several embodiments, there are provided engineered NK cells,engineered T cells, and/or mixed populations of NK cells and T cellsthat express one or more of the humanized CD19-directed chimeric antigenreceptors provided for herein.

Also provided are methods for treating cancer in a subject comprisingadministering to a subject having cancer the engineered NK and/or Tcells expressing chimeric antigen receptors as disclosed herein. Alsoprovided for are the use of the polynucleotides provided for herein forthe treatment of cancer as well as use of the polynucleotides providedfor herein in the manufacture of a medicament for the treatment ofcancer.

Also provided for herein, in several embodiments, is a polynucleotideencoding a CD19-directed chimeric antigen receptor, the chimeric antigenreceptor comprising an extracellular anti-CD19 binding moiety, whereinthe anti-CD19 binding moiety comprises a scFv, a transmembrane domain,and an intracellular signaling domain, wherein the intracellularsignaling domain comprises a CD28 co-stimulatory domain and a CD3 zetasignaling domain.

Also provided for herein, in several embodiments, is a polynucleotideencoding a CD19-directed chimeric antigen receptor, the chimeric antigenreceptor comprising an extracellular anti-CD19 binding moiety, whereinthe anti-CD19 binding moiety comprises a scFv, a hinge, wherein thehinge is a CD8 alpha hinge, a transmembrane domain, and an intracellularsignaling domain, wherein the intracellular signaling domain comprises aCD3 zeta ITAM.

Also provided for herein, in several embodiments, is a polynucleotideencoding a CD19-directed chimeric antigen receptor, the chimeric antigenreceptor comprising an extracellular anti-CD19 binding moiety, whereinthe anti-CD19 binding moiety comprises a variable heavy chain of a scFvor a variable light chain of a scFv, a hinge, wherein the hinge is a CD8alpha hinge, a transmembrane domain, wherein the transmembrane domaincomprises a CD8 alpha transmembrane domain, and an intracellularsignaling domain, wherein the intracellular signaling domain comprises aCD3 zeta ITAM.

In several embodiments, the transmembrane domain comprises a CD8 alphatransmembrane domain. In several embodiments, the transmembrane domaincomprises an NKG2D transmembrane domain. In several embodiments, thetransmembrane domain comprises a CD28 transmembrane domain.

In several embodiments the intracellular signaling domain comprises orfurther comprises a CD28 signaling domain. In several embodiments, theintracellular signaling domain comprises or further comprises a 4-1 BBsignaling domain. In several embodiments, the intracellular signalingdomain comprises an or further comprises OX40 domain. In severalembodiments, the intracellular signaling domain comprises or furthercomprises a 4-1 BB signaling domain. In several embodiments, theintracellular signaling domain comprises or further comprises a domainselected from ICOS, CD70, CD161, CD40L, CD44, and combinations thereof.

In several embodiments, the polynucleotide also encodes a truncatedepidermal growth factor receptor (EGFRt). In several embodiments, theEGFRt is expressed in a cell as a soluble factor. In severalembodiments, the EGFRt is expressed in a membrane bound form. In severalembodiments, the EGFRt operates to provide a “suicide switch” functionin the engineered NK cells. In several embodiments, the polynucleotidealso encodes membrane-bound interleukin-15 (mbIL15). Also provided forherein are engineered immune cells (e.g., NK or T cells, or mixturesthereof) that express a CD19-directed chimeric antigen receptor encodedby a polynucleotide disclosed herein. Further provided are methods fortreating cancer in a subject comprising administering to a subjecthaving cancer engineered immune cells expressing the chimeric antigenreceptors disclosed herein. In several embodiments, there is providedthe use of the polynucleotides disclosed herein in the treatment ofcancer and/or in the manufacture of a medicament for the treatment ofcancer.

In several embodiments, the anti-CD19 binding moiety comprises a heavychain variable (VH) domain and a light chain variable (VL) domain. Inseveral embodiments, the VH domain has at least 95% (e.g., 95, 96, 97,98, or 99%) identity to the VH domain amino acid sequence set forth inSEQ ID NO: 33. In several embodiments, the VL domain has at least 95%(e.g., 95, 96, 97, 98, or 99%) identity to the VL domain amino acidsequence set forth in SEQ ID NO: 32. In several embodiments, theanti-CD19 binding moiety is derived from the VH and/or VL sequences ofSEQ ID NO: 33 or 32. For example, in several embodiments, the VH and VLsequences for SEQ ID NO: 33 and/or 32 are subject to a humanizationcampaign and therefore are expressed more readily and/or lessimmunogenic when administered to human subjects. Thus, in severalembodiments, the anti-CD19 binding moiety does not comprise SEQ ID NO:32 and/or SEQ ID NO: 33. In several embodiments, the anti-CD19 bindingmoiety comprises a scFv that targets CD19 wherein the scFv comprises aheavy chain variable region comprising the sequence of SEQ ID NO. 35 ora sequence at least 95% (e.g., 95, 96, 97, 98, or 99%) identical to SEQID NO: 35. In several embodiments, the anti-CD19 binding moietycomprises an scFv that targets CD19 comprises a light chain variableregion comprising the sequence of SEQ ID NO. 36 or a sequence at least95% identical (e.g., 95, 96, 97, 98, or 99%) to SEQ ID NO: 36. Inseveral embodiments, the anti-CD19 binding moiety comprises a lightchain CDR comprising a first, second and third complementaritydetermining region (LC CDR1, LC CDR2, and LC CDR3, respectively) and/ora heavy chain CDR comprising a first, second and third complementaritydetermining region (HC CDR1, HC CDR2, and HC CDR3, respectively).Depending on the embodiment, various combinations of the LC CDRs and HCCDRs are used. For example, in one embodiment the anti-CD19 bindingmoiety comprises LC CDR1, LC CDR3, HC CD2, and HC, CDR3. Othercombinations are used in some embodiments. In several embodiments, theLC CDR1 comprises the sequence of SEQ ID NO. 37 or a sequence at leastabout 95% homologous to the sequence of SEQ NO. 37. In severalembodiments, the LC CDR2 comprises the sequence of SEQ ID NO. 38 or a ora sequence at least about 95% (e.g., 96, 97, 98, or 99%) homologous tothe sequence of SEQ NO. 38. In several embodiments, the LC CDR3comprises the sequence of SEQ ID NO. 39 or a sequence at least about 95%homologous to the sequence of SEQ NO. 39. In several embodiments, the HCCDR1 comprises the sequence of SEQ ID NO. 40 or a sequence at leastabout 95% homologous to the sequence of SEQ NO. 40. In severalembodiments, the HC CDR2 comprises the sequence of SEQ ID NO. 41, 42, or43 or a sequence at least about 95% homologous to the sequence of SEQNO. 41, 42, or 43. In several embodiments, the HC CDR3 comprises thesequence of SEQ ID NO. 44 or a sequence at least about 95% (e.g., 96,97, 98, 99 or 99%) homologous to the sequence of SEQ NO. 44.

In several embodiments, there is also provided an anti-CD19 bindingmoiety that comprises a light chain variable region (VL) and a heavychain variable region (HL), the VL region comprising a first, second andthird complementarity determining region (VL CDR1, VL CDR2, and VL CDR3,respectively and the VH region comprising a first, second and thirdcomplementarity determining region (VH CDR1, VH CDR2, and VH CDR3,respectively. In several embodiments, the VL region comprises thesequence of SEQ ID NO. 45, 46, 47, or 48 or a sequence at least about95% (e.g., 96, 97, 98, 99 or 99%) homologous to the sequence of SEQ NO.45, 46, 47, or 48. In several embodiments, the VH region comprises thesequence of SEQ ID NO. 49, 50, 51 or 52 or a sequence at least about 95%(e.g., 96, 97, 98, 99 or 99%) homologous to the sequence of SEQ NO. 49,50, 51 or 52.

In several embodiments, there is also provided an anti-CD19 bindingmoiety that comprises a light chain CDR comprising a first, second andthird complementarity determining region (LC CDR1, LC CDR2, and LC CDR3,respectively. In several embodiments, the anti-CD19 binding moietyfurther comprises a heavy chain CDR comprising a first, second and thirdcomplementarity determining region (HC CDR1, HC CDR2, and HC CDR3,respectively. In several embodiments, the LC CDR1 comprises the sequenceof SEQ ID NO. 53 or a sequence at least about 95% homologous to thesequence of SEQ NO. 53. In several embodiments, the LC CDR2 comprisesthe sequence of SEQ ID NO. 54 or a sequence at least about 95%homologous to the sequence of SEQ NO. 54. In several embodiments, the LCCDR3 comprises the sequence of SEQ ID NO. 55 or a sequence at leastabout 95% homologous to the sequence of SEQ NO. 55. In severalembodiments, the HC CDR1 comprises the sequence of SEQ ID NO. 56 or asequence at least about 95% homologous to the sequence of SEQ NO. 56. Inseveral embodiments, the HC CDR2 comprises the sequence of SEQ ID NO. 57or a sequence at least about 95% homologous to the sequence of SEQ NO.57. In several embodiments, the HC CDR3 comprises the sequence of SEQ IDNO. 58 or a sequence at least about 95% homologous to the sequence ofSEQ NO. 58. In several embodiments, the anti-CD19 binding moiety (andthus the resultant CAR) is engineered to not include certain sequences,such as, for example, those that may cause increased risk ofimmunogenicity and/or side effects, such as cytokine release syndrome.Thus, according to several embodiments, the anti-CD19 binding moietydoes not comprise one or more of SEQ ID NO: 37, SEQ ID NO: 38, SEQ IDNO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48,SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52. SEQ ID NO:53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 32, or SEQ ID NO: 33.

In several embodiments, the intracellular signaling domain of thechimeric receptor comprises an OX40 subdomain. In several embodiments,the intracellular signaling domain further comprises a CD3zetasubdomain. In several embodiments, the OX40 subdomain comprises theamino acid sequence of SEQ ID NO: 16 (or a sequence at least about 95%homologous to the sequence of SEQ ID NO. 16) and the CD3zeta subdomaincomprises the amino acid sequence of SEQ ID NO: 8 (or a sequence atleast about 95% homologous to the sequence of SEQ ID NO: 8).

In several embodiments, the hinge domain comprises a CD8a hinge domain.In several embodiments, the CD8a hinge domain, comprises the amino acidsequence of SEQ ID NO: 2 or a sequence at least about 95% homologous tothe sequence of SEQ ID NO: 2).

In several embodiments, the immune cell also expresses membrane-boundinterleukin-15 (mbIL15). In several embodiments, the mbIL15 comprisesthe amino acid sequence of SEQ ID NO: 12 or a sequence at least about95% homologous to the sequence of SEQ ID NO: 12.

In several embodiments, wherein the chimeric receptor further comprisesan extracellular domain of an NKG2D receptor. In several embodiments,the immune cell expresses a second chimeric receptor comprising anextracellular domain of an NKG2D receptor, a transmembrane domain, acytotoxic signaling complex and optionally, mbIL15. In severalembodiments, the extracellular domain of the NKG2D receptor comprises afunctional fragment of NKG2D comprising the amino acid sequence of SEQID NO: 26 or a sequence at least about 95% homologous to the sequence ofSEQ ID NO: 26. In various embodiments, the immune cell engineered toexpress the chimeric antigen receptor and/or chimeric receptorsdisclosed herein is an NK cell. In some embodiments, T cells are used.In several embodiments, combinations of NK and T cells (and/or otherimmune cells) are used.

In several embodiments, there are provided herein methods of treatingcancer in a subject comprising administering to the subject having anengineered immune cell targeting CD19 as disclosed herein. Also providedfor herein is the use of an immune cell targeting CD19 as disclosedherein for the treatment of cancer. Likewise, there is provided forherein the use of an immune cell targeting CD19 as disclosed herein inthe preparation of a medicament for the treatment of cancer. In severalembodiments, the cancer treated is acute lymphocytic leukemia.

Some embodiments of the methods and compositions described herein relateto an immune cell. In some embodiments, the immune cell expresses aCD19-directed chimeric receptor comprising an extracellular anti-CD19moiety, a hinge and/or transmembrane domain, and/or an intracellularsignaling domain. In some embodiments, the immune cell is a naturalkiller (NK) cell. In some embodiments, the immune cell is a T cell.

In some embodiments, the hinge domain comprises a CD8a hinge domain. Insome embodiments, the hinge domain comprises an Ig4 SH domain.

In some embodiments, the transmembrane domain comprises a CD8atransmembrane domain. In some embodiments, the transmembrane domaincomprises a CD28 transmembrane domain. In some embodiments, thetransmembrane domain comprises a CD3 transmembrane domain.

In some embodiments, the signaling domain comprises an OX40 signalingdomain. In some embodiments, the signaling domain comprises a 4-1 BBsignaling domain. In some embodiments, the signaling domain comprises aCD28 signaling domain. In some embodiments, the signaling domaincomprises an NKp80 signaling domain. In some embodiments, the signalingdomain comprises a CD16 IC signaling domain. In some embodiments, thesignaling domain comprises a CD3zeta or CD3ζ ITAM signaling domain. Insome embodiments, the signaling domain comprises an mbIL-15 signalingdomain. In some embodiments, the signaling domain comprises a 2Acleavage domain. In some embodiments, the mIL-15 signaling domain isseparated from the rest or another portion of the CD19-directed chimericreceptor by a 2A cleavage domain.

Some embodiments relate to a method comprising administering an immunecell as described herein to a subject in need. In some embodiments, thesubject has cancer. In some embodiments, the administration treats,inhibits, or prevents progression of the cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A includes depictions of non-limiting examples of CD19-directedchimeric receptors.

FIG. 1B includes depictions of additional non-limiting examples ofCD19-directed chimeric receptors.

FIG. 2 also includes depictions of non-limiting examples ofCD19-directed chimeric receptors.

FIG. 3A also includes depictions of non-limiting examples ofCD19-directed chimeric receptors.

FIG. 3B also includes depictions of non-limiting examples ofCD19-directed chimeric receptors.

FIG. 3C also includes depictions of non-limiting examples ofCD19-directed chimeric receptors.

FIG. 3D also includes depictions of non-limiting examples ofCD19-directed chimeric receptors comprising humanized CD19 bindingdomains.

FIG. 3E also includes depictions of non-limiting examples ofCD19-directed chimeric receptors comprising humanized CD19 bindingdomains.

FIG. 3F also includes depictions of non-limiting examples ofCD19-directed chimeric receptors comprising humanized CD19 bindingdomains.

FIG. 3G also includes depictions of non-limiting examples ofCD19-directed chimeric receptors comprising humanized CD19 bindingdomains without a tag sequence.

FIG. 3H also includes depictions of non-limiting examples ofCD19-directed chimeric receptors comprising humanized CD19 bindingdomains without a tag sequence.

FIG. 3I also includes depictions of non-limiting examples ofCD19-directed chimeric receptors comprising humanized CD19 bindingdomains without a tag sequence.

FIG. 4 depicts a schematic of a non-limiting NK cell expansion protocol.

FIG. 5 shows a schematic of an experimental protocol assessing theeffectiveness of a CD19-directed chimeric antigen receptor in accordancewith several embodiments disclosed herein.

FIG. 6A depicts in vivo data related to the anti-tumor effect of variousnon-limiting CD19-directed chimeric receptors.

FIG. 6B depicts summary data related to the anti-tumor effect of variousnon-limiting CD19-directed chimeric receptors.

FIG. 7 depicts data related to the expression of various CD19-directedchimeric receptor constructs by NK cells.

FIG. 8A depicts raw fluorescence data (mean fluorescence intensity, MFI)related to the expression of selected CD19-directed chimeric receptors.

FIG. 8B depicts data related to the expression of selected CD19-directedchimeric receptors, displayed as percent of NK cells expressing theindicated receptor.

FIGS. 9A-9D depicts data related to the cytotoxicity (against Nalm6 orRaji cells) for the indicated CD19-directed chimeric receptors.

FIG. 9E depicts summary data related to the cytotoxicity (against Nalm6cells) for the indicated CD19-directed chimeric receptors at variouseffector:target ratios.

FIG. 9F depicts summary data related to the cytotoxicity (against Rajicells) for the indicated CD19-directed chimeric receptors at variouseffector:target ratios.

FIG. 10A depicts data related to the degree of enhanced cytotoxicity(against Nalm6 cells) for the indicated CD19-directed chimeric receptorsat 7 days post transduction.

FIG. 10B depicts data related to the degree of enhanced cytotoxicity(against Raji cells) for the indicated CD19-directed chimeric receptorsat 7 days post transduction.

FIG. 100 depicts data related to the degree of enhanced cytotoxicity(against Nalm6 cells) for the indicated CD19-directed chimeric receptorsat 14 days post transduction.

FIG. 10D depicts data related to the degree of enhanced cytotoxicity(against Raji cells) for the indicated CD19-directed chimeric receptorsat 14 days post transduction.

FIGS. 11A-11E depict data related to cytokine release by NK cellsexpressing various CD19-directed chimeric receptors when co-culturedwith Nalm6 cells. FIG. 11A depicts Granzyme B release. FIG. 11B depictsperforin release. FIG. 11C depicts TNF-alpha release. FIG. 11D depictsGM-CSF release. FIG. 11E depicts interferon gamma release.

FIGS. 12A-12E depict data related to cytokine release by NK cellsexpressing various CD19-directed chimeric receptors when co-culturedwith Raji cells. FIG. 12A depicts Granzyme B release. FIG. 12B depictsperforin release. FIG. 12C depicts TNF-alpha release. FIG. 12D depictsGM-CSF release. FIG. 12E depicts interferon gamma release.

FIG. 13 depicts in vivo imaging data related to tumor burden over timein mice treated with PBS, non-transduced NK cells, or NK cellsexpressing the indicated CD19-directed chimeric receptors, with theindicated number of engineered NK cells administered (M=million cells).

FIG. 14A shows raw data related to the detected fluorescent signal fromthe in vivo data shown in FIG. 13, with data being tracked for 25 dayspost-administration of Nalm6 cells.

FIG. 14B shows the data from FIG. 14A on a logarithmic scale.

FIG. 14C shows data related to CD3 expression by the indicated cellsexpressing the indicated constructs.

FIG. 14D shows data related to CD56 expression by the indicated cellsexpressing the indicated constructs.

FIG. 14E shows data related to GFP-expressing tumor cells when contactedwith the indicated cells expressing the indicated constructs.

FIG. 14F shows the data related to CD19-expressing tumor cells whencontacts with the indicated cells expressing the indicated constructs.

FIG. 15 depicts data related to selected functional characteristics ofselected humanized anti-CD19 CAR constructs.

FIG. 16 depicts data related to the expression of various humanizedanti-CD19 CAR constructs disclosed in embodiments herein.

FIGS. 17A-17E show cytotoxicity data. 17A shows data related to thecytotoxicity of NK cells from a first donor engineered to expressselected humanized CD19 CAR constructs disclosed herein against Nalm6cells. 17A shows data related to the cytotoxicity of NK cells from afirst donor engineered to express selected humanized CD19 CAR constructsdisclosed herein against Raji cells. 17C shows data related to thecytotoxicity of NK cells from a second donor engineered to expressselected humanized CD19 CAR constructs disclosed herein against Nalm6cells. 17D shows data related to the cytotoxicity of NK cells from athird donor engineered to express selected humanized CD19 CAR constructsdisclosed herein against Raji cells. 17E shows data related to thecytotoxicity of NK cells from a third donor engineered to expressselected humanized CD19 CAR constructs disclosed herein against Nalm6cells.

FIG. 18 shows summary (from 3 donors) expression data for NK cellsexpressing the indicated non-limiting anti-CD19 CAR constructs at 3 dayspost-transduction.

FIGS. 19A-19D show cytotoxicity rechallenge data. FIG. 19A shows datarelated to the cytotoxicity of NK cells from a first donor engineered toexpress selected humanized CD19 CAR constructs disclosed herein againstRaji cells with addition of the Raji cells to the NK cell culture at day7 and again at day 14. FIG. 19B shows data related to the cytotoxicityof NK cells from a first donor engineered to express selected humanizedCD19 CAR constructs disclosed herein against Nalm6 cells with additionof the Nalm6 cells to the NK cell culture at day 7 and again at day 14.FIG. 19C shows data related to the cytotoxicity of NK cells from asecond donor engineered to express selected humanized CD19 CARconstructs disclosed herein against Raji cells with addition of the Rajicells to the NK cell culture at day 7 and again at day 14. FIG. 19Dshows data related to the cytotoxicity of NK cells from a second donorengineered to express selected humanized CD19 CAR constructs disclosedherein against Nalm6 cells with addition of the Nalm6 cells to the NKcell culture at day 7 and again at day 14.

FIGS. 20A-20B show expression data. FIG. 20A shows fluorescence datafrom expression of non-limiting examples of humanized anti-CD19 CARconstructs from two donors 10 days post-transduction. FIG. 20B showsdata related to the percentage of cells expressing CD19 (indicative ofthe efficiency of expression of the given anti-CD19 CAR.

FIGS. 21A-21B show cytotoxicity data. FIG. 21A shows data related to thecytotoxicity of NK cells (from the first of the two donors of FIG. 20)engineered to express selected humanized CD19 CAR constructs disclosedherein against Raji cells with addition of the Raji cells to the NK cellculture at day 7 and again at day 14. FIG. 21B shows data related to thecytotoxicity of NK cells (from the second of the two donors of FIG. 20)engineered to express selected humanized CD19 CAR constructs disclosedherein against Raji cells with addition of the Raji cells to the NK cellculture at day 7 and again at day 14.

FIGS. 22A-22E show data related to cytokine release by NK cellsexpressing various CD19-directed chimeric receptors when co-culturedwith Raji cells. FIG. 22A shows interferon gamma release. FIG. 22B showsGM-CSF release. FIG. 22C shows tumor necrosis factor release. FIG. 22Dshows perforin release. FIG. 22E shows granzyme release.

FIGS. 23A-23D show data related to NK cell survival. FIG. 23A shows theanalysis of survival of NK cells from a first donor engineered toexpress the indicated non-limiting embodiments of anti-CD19 CARconstructs at days 7, 13, 19, and 26 post-transduction. FIG. 23B showsthe analysis of survival of NK cells from a second donor engineered toexpress the indicated non-limiting embodiments of anti-CD19 CARconstructs at days 7, 13, 19, and 26 post-transduction. FIG. 23C showsthe analysis of survival of NK cells from a third donor engineered toexpress the indicated non-limiting embodiments of anti-CD19 CARconstructs at days 11, 19, and 26 post-transduction. FIG. 23D shows theanalysis of survival of NK cells from a fourth donor engineered toexpress the indicated non-limiting embodiments of anti-CD19 CARconstructs at days 11, 19, and 26 post-transduction. For eachexperiment, media was changed twice per week.

FIG. 24 shows data related to the percent of NK cells expressing theindicated non-limiting embodiments of anti-CD19 CAR constructs at 11days post-transduction.

FIGS. 25A-25I show data related to the expression of CD19 by NK cellstransduced with various non-limiting embodiments of anti-CD19 CARconstructs disclosed herein. FIG. 25A shows GFP transduced NK cells as acontrol. FIG. 25B shows CD19 expression in NK cells transduced withNK19-1. FIG. 25C shows CD19 expression in NK cells transduced withNK19H-1. FIG. 25D shows CD19 expression in NK cells transduced withNK19H-2. FIG. 25E shows CD19 expression in NK cells transduced withNK19H-3. FIG. 25F shows CD19 expression in NK cells transduced withNK19H-4.

FIG. 25G shows CD19 expression in NK cells transduced with NK19H-5. FIG.25H shows CD19 expression in NK cells transduced with NK19H-11. FIG. 25Ishows CD19 expression in NK cells transduced with NK19H-12.

FIGS. 26A-26D show cytotoxicity data. FIG. 26A shows data related to thecytotoxicity of NK cells (from a first donor) engineered to expressselected humanized CD19 CAR constructs disclosed herein against Rajicells with addition of the Raji cells to the NK cell culture at day 7and again at day 14.

FIG. 26B shows data related to the cytotoxicity of NK cells (from thefirst donor) engineered to express selected humanized CD19 CARconstructs disclosed herein against Nalm6 cells with addition of theNalm6 cells to the NK cell culture at day 7 and again at day 14. FIG.26C shows data related to the cytotoxicity of NK cells (from a seconddonor) engineered to express selected humanized CD19 CAR constructsdisclosed herein against Raji cells with addition of the Raji cells tothe NK cell culture at day 7 and again at day 14. FIG. 26D shows datarelated to the cytotoxicity of NK cells (from the second donor)engineered to express selected humanized CD19 CAR constructs disclosedherein against Nalm6 cells with addition of the Nalm6 cells to the NKcell culture at day 7 and again at day 14.

FIGS. 27A-27B show data related to CD19 expression by engineered NKcells. FIG. 27A shows flow cytometry data for NK cells from a donor thatwere engineered to express various non-limiting anti-CD19 CAR constructsdisclosed herein. FIG. 27B shows corresponding data from NK cellsisolated from an additional donor.

FIGS. 28A-28B show cytotoxicity data. FIG. 28A shows the Raji cell countover time when exposed (at 1:1 E:T ratio) to NK cells (from the firstdonor of FIG. 27) expressing the non-limiting examples of anti-CD19 CARconstructs indicated. FIG. 28B shows the Raji cell count over time whenexposed (at 1:1 E:T ratio) to NK cells (from the second donor of FIG.27) expressing the non-limiting examples of anti-CD19 CAR constructsindicated.

FIGS. 29A-29E show data related to various humanized CD19-directed CARconstructs and their efficacy in an in vivo model. FIG. 29A shows aschematic depiction of an experimental protocol for assessing theeffectiveness of CD19-directed CAR constructs in vivo. FIG. 29B shows invivo bioluminescent imaging of mice having been administered NALM6 tumorcells and treated with the indicated construct. FIG. 29C shows a linegraph of the bioluminescent data collected from FIG. 29B. FIG. 29D is asurvival curve showing the days that animals in each treatment groupsurvived. FIG. 29E shows the relative expression level of the indicatedconstructs by NK cells, as measured by the MFI of a tag included in theCD19 CAR construct.

FIGS. 30A-30F relate to expression of the indicated CD19-directed CARconstructs. FIG. 30A shows data related to the expression of theindicated constructs as a percentage of all CD56 positive cells in ablood sample from mice 15 days post-NK cell administration (e.g., as perthe schematic in FIG. 29A). FIG. 30B shows data related to expression ofthe indicated constructs as a percentage of cells that are both CD56positive and express a Flag tag (as part of the CD19 CAR construct),also at 15 days post-NK cell administration. FIG. 30C shows data relatedto detection of GFP+ tumor cells in samples from animals treated withthe indicated constructs, also at day 15 post-NK cell administration.FIGS. 30D, 30E, and 30F show corresponding data at 32 days post-NK celladministration.

FIGS. 31A-31B show data related to the expression of non-tagged CD19 CARconstructs. As discussed herein, in several embodiments the CD19 CARconstructs comprise a Flag, or other tag, for detection purposes.However, all constructs disclosed herein with a tag are also includedherein without a tag. FIG. 31A shows data related to the expressing ofselected non-flag tagged humanized anti CD19 CAR constructs over time.FIG. 31B shows corresponding data in terms of the detected meanfluorescent intensity for the indicated constructs.

FIGS. 32A-32D show data related to an in vitro re-challenge experimentin which a first population of tumor cells were co-cultured with NKcells expressing the indicated constructs and the NK cells were“rechallenged” with an additional bolus of tumor cells. FIG. 32A showsdata at 10 days after inception of co-culture with Raji cells. FIG. 32Bshows data related to Raji at the final time point, 14 days. FIG. 32Cshows data related to Nalm6 cells at 10 days. FIG. 32D shows datarelated to Nalm6 cells at 14 days.

FIGS. 33A-33J relate to evaluation of cytokine production by NK cellsexpressing the indicated constructs. FIG. 33A shows expression ofinterferon gamma by NK cells expressing the indicated constructs afterco-culturing with Raji cells. FIG. 33B shows expression of GM-CSF by NKcells expressing the indicated constructs after co-culturing with Rajicells. FIG. 33C shows expression of tumor necrosis factor alpha by NKcells expressing the indicated constructs after co-culturing with Rajicells. FIG. 33D shows expression of perforin by NK cells expressing theindicated constructs after co-culturing with Raji cells. FIG. 33E showsexpression of granzyme B by NK cells expressing the indicated constructsafter co-culturing with Raji cells. FIG. 33F shows expression ofinterferon gamma by NK cells expressing the indicated constructs afterco-culturing with Nalm6 cells. FIG. 33G shows expression of GM-CSF by NKcells expressing the indicated constructs after co-culturing withNalm6cells. FIG. 33H shows expression of tumor necrosis factor alpha byNK cells expressing the indicated constructs after co-culturing withNalm6cells. FIG. 33I shows expression of perforin by NK cells expressingthe indicated constructs after co-culturing with Nalm6 cells. FIG. 33Jshows expression of granzyme B by NK cells expressing the indicatedconstructs after co-culturing with Nalm6 cells.

FIG. 34 shows data related to the viability of NK cells expressinghumanized non-tagged CD19 CAR constructs over four weekspost-transduction.

FIGS. 35A-35D show data related to various humanized, non-tagged,CD19-directed CAR constructs and their efficacy in an in vivo model.FIG. 35A shows a schematic depiction of an experimental protocol forassessing the effectiveness of humanized, non-tagged, CD19-directed CARconstructs in vivo. FIG. 35B shows in vivo bioluminescent imaging ofmice having been administered NALM6 tumor cells and treated with theindicated construct. FIG. 35C shows a line graph of the bioluminescentdata collected from FIG. 35B. FIG. 35D shows the relative expressionlevel of the indicated constructs by NK cells, as measured by detectionof a CD19-Fc fusion protein that binds to the CD19 CAR construct.

FIGS. 36A-36F relate to expression of the indicated CD19-directed CARconstructs. FIG. 36A shows data related to the expression of theindicated constructs as a percentage of all CD56 positive cells in ablood sample from mice 13 days post-NK cell administration (e.g., as perthe schematic in FIG. 35A). FIG. 36B shows data related to detection ofCD19+ tumor cells in samples from animals treated with the indicatedconstructs, also at 13 days post-NK cell administration. FIG. 36C showsdata related to detection of GFP+ tumor cells in samples from animalstreated with the indicated constructs, also at 13 days post-NK celladministration. FIGS. 36D, 36E, and 36F show corresponding data at 27days post-NK cell administration.

FIGS. 37A-37C relate to the in vivo efficacy of various CD19-directedCAR according to embodiments disclosed herein. FIG. 37A shows aschematic depiction of an experimental protocol for assessing theeffectiveness of humanized, NK cells expressing various CD19-directedCAR constructs in vivo. The various experimental groups tested are asindicated. For cells with an “IL12/IL18” designation, the cells wereexpanded in the presence of soluble IL12 and/or IL18, as described in inU.S. Provisional Patent Application No. 62/881,311, filed Jul. 31, 2019and Application No. 62/932,342, filed Nov. 7, 2019, each of which isincorporated in its entirety by reference herein. FIGS. 37B and 37C showbioluminescence data from animals dosed with Nalm6 tumor cells andtreated with the indicated construct.

FIGS. 38A-38J show graphical depictions of the bioluminescence data fromFIG. 37B. FIG. 38A shows bioluminescence (as photon/second flux) fromanimals receiving untransduced NK cells. FIG. 38B shows flux measured inanimals receiving PBS as a vehicle. FIG. 38C shows flux measured inanimals receiving previously frozen NK cells expressing the NK19 NF2 CAR(as a non-limiting example of a CAR). FIG. 38D shows flux measured inanimals receiving previously frozen NK cells expressing the NK19 NF2 CAR(as a non-limiting example of a CAR) expanded using IL12 and/or IL18.FIG. 38E and FIG. 38F show flux measured in animals receiving fresh NKcells expressing the NK19 NF2 CAR (as a non-limiting example of a CAR).FIG. 38G and FIG. 38H show flux measured in animals receiving previouslyfresh NK cells expressing the NK19 NF2 CAR (as a non-limiting example ofa CAR) expanded using IL12 and/or IL18. FIG. 38I shows a line graphdepicting the bioluminescence measured in the various groups over thefirst 30 days post-tumor inoculation. FIG. 38J shows a line graphdepicting the bioluminescence measured in the various groups over thefirst 56 days post-tumor inoculation.

FIG. 39 shows data related to the body mass of mice over time whenreceiving the indicated therapy.

DETAILED DESCRIPTION

Some embodiments of the methods and compositions provided herein relateto CD19-directed chimeric receptors. In some embodiments, the receptorsare expressed on a cell as described herein. Some embodiments includemethods of use of the compositions or cells in immunotherapy.

The term “anticancer effect” refers to a biological effect which can bemanifested by various means, including but not limited to, a decrease intumor volume, a decrease in the number of cancer cells, a decrease inthe number of metastases, an increase in life expectancy, decrease incancer cell proliferation, decrease in cancer cell survival, oramelioration of various physiological symptoms associated with thecancerous condition. An “anticancer effect” can also be manifested bythe ability of the SIRs in prevention of the occurrence of cancer in thefirst place.

Cell Types

Some embodiments of the methods and compositions provided herein relateto a cell such as an immune cell. For example, an immune cell may beengineered to include a chimeric receptor such as a CD19-directedchimeric receptor, or engineered to include a nucleic acid encoding saidchimeric receptor as described herein.

Traditional anti-cancer therapies relied on a surgical approach,radiation therapy, chemotherapy, or combinations of these methods. Asresearch led to a greater understanding of some of the mechanisms ofcertain cancers, this knowledge was leveraged to develop targeted cancertherapies. Targeted therapy is a cancer treatment that employs certaindrugs that target specific genes or proteins found in cancer cells orcells supporting cancer growth, (like blood vessel cells) to reduce orarrest cancer cell growth. More recently, genetic engineering hasenabled approaches to be developed that harness certain aspects of theimmune system to fight cancers. In some cases, a patient's own immunecells are modified to specifically eradicate that patient's type ofcancer. Various types of immune cells can be used, such as T cells orNatural Killer (NK cells), as described in more detail below.

To facilitate cancer immunotherapies, there are provided for hereinpolynucleotides, polypeptides, and vectors that encode chimeric antigenreceptors (CAR) that comprise a target binding moiety (e.g., anextracellular binder of a ligand, or a CD19-directed chimeric receptor,expressed by a cancer cell) and a cytotoxic signaling complex. Forexample, some embodiments include a polynucleotide, polypeptide, orvector that encodes a CD19-directed chimeric receptor to facilitatetargeting of an immune cell to a cancer and exerting cytotoxic effectson the cancer cell. Also provided are engineered immune cells (e.g., Tcells or NK cells) expressing such CARs. There are also provided herein,in several embodiments, polynucleotides, polypeptides, and vectors thatencode a construct comprising an extracellular domain comprising two ormore subdomains, e.g., first CD19-targeting subdomain comprising a CD19binding moiety as disclosed herein and a second subdomain comprising aC-type lectin-like receptor and a cytotoxic signaling complex. Alsoprovided are engineered immune cells (e.g., T cells or NK cells)expressing such bi-specific constructs. Engineered immune cells (e.g., Tcells or NK cells) expressing multi-specific constructs and/or havingthe ability to bind a plurality of target markers are also provided.Methods of treating cancer and other uses of such cells for cancerimmunotherapy are also provided for herein.

Engineered Cells for Immunotherapy

In several embodiments, cells of the immune system are engineered tohave enhanced cytotoxic effects against target cells, such as tumorcells. For example, a cell of the immune system may be engineered toinclude a CD19-directed chimeric receptor as described herein. Inseveral embodiments, white blood cells or leukocytes, are used, sincetheir native function is to defend the body against growth of abnormalcells and infectious disease. There are a variety of types of whitebloods cells that serve specific roles in the human immune system, andare therefore a preferred starting point for the engineering of cellsdisclosed herein. White blood cells include granulocytes andagranulocytes (presence or absence of granules in the cytoplasm,respectively). Granulocytes include basophils, eosinophils, neutrophils,and mast cells. Agranulocytes include lymphocytes and monocytes. Cellssuch as those that follow or are otherwise described herein may beengineered to include a chimeric receptor such as a CD19-directedchimeric receptor, or a nucleic acid encoding the chimeric receptorand/or engineered to co-express a membrane-bound interleukin 15 (mbIL15)co-stimulatory domain.

Monocytes for Immunotherapy

Monocytes are a subtype of leukocyte. Monocytes can differentiate intomacrophages and myeloid lineage dendritic cells. Monocytes areassociated with the adaptive immune system and serve the main functionsof phagocytosis, antigen presentation, and cytokine production.Phagocytosis is the process of uptake cellular material, or entirecells, followed by digestion and destruction of the engulfed cellularmaterial. In several embodiments, monocytes are used in connection withone or more additional engineered cells as disclosed herein. Someembodiments of the methods and compositions described herein relate to amonocyte that includes a CD19-directed chimeric receptor, or a nucleicacid encoding the CD19-directed chimeric receptor. Several embodimentsof the methods and compositions disclosed herein relate to monocytesengineered to express a CD19-directed chimeric receptor and amembrane-bound interleukin 15 (mbIL15) co-stimulatory domain.

Lymphocytes for Immunotherapy

Lymphocytes, the other primary sub-type of leukocyte include T cells(cell-mediated, cytotoxic adaptive immunity), natural killer cells(cell-mediated, cytotoxic innate immunity), and B cells (humoral,antibody-driven adaptive immunity). While B cells are engineeredaccording to several embodiments, disclosed herein, several embodimentsalso relate to engineered T cells or engineered NK cells (mixtures of Tcells and NK cells are used in some embodiments). Some embodiments ofthe methods and compositions described herein relate to a lymphocytethat includes a CD19-directed chimeric receptor, or a nucleic acidencoding the CD19-directed chimeric receptor. Several embodiments of themethods and compositions disclosed herein relate to lymphocytesengineered to express a CD19-directed chimeric receptor and amembrane-bound interleukin 15 (mbIL15) co-stimulatory domain.

T Cells for Immunotherapy

T cells are distinguishable from other lymphocytes sub-types (e.g., Bcells or NK cells) based on the presence of a T-cell receptor on thecell surface. T cells can be divided into various different subtypes,including effector T cells, helper T cells, cytotoxic T cells, memory Tcells, regulatory T cells, natural killer T cell, mucosal associatedinvariant T cells and gamma delta T cells. In some embodiments, aspecific subtype of T cell is engineered. In some embodiments, a mixedpool of T cell subtypes is engineered. In some embodiments, there is nospecific selection of a type of T cells to be engineered to express thecytotoxic receptor complexes disclosed herein. In several embodiments,specific techniques, such as use of cytokine stimulation are used toenhance expansion/collection of T cells with a specific marker profile.For example, in several embodiments, activation of certain human Tcells, e.g. CD4+ T cells, CD8+ T cells is achieved through use of CD3and/or CD28 as stimulatory molecules. In several embodiments, there isprovided a method of treating or preventing cancer or an infectiousdisease, comprising administering a therapeutically effective amount ofT cells expressing the cytotoxic receptor complex and/or a homing moietyas described herein. In several embodiments, the engineered T cells areautologous cells, while in some embodiments, the T cells are allogeneiccells. Some embodiments of the methods and compositions described hereinrelate to a T cell that includes a CD19-directed chimeric receptor, or anucleic acid encoding the CD19-directed chimeric receptor. Severalembodiments of the methods and compositions disclosed herein relate toT-cells engineered to express a CD19-directed chimeric receptor and amembrane-bound interleukin 15 (mbIL15) co-stimulatory domain.

NK Cells for Immunotherapy

In several embodiments, there is provided a method of treating orpreventing cancer or an infectious disease, comprising administering atherapeutically effective amount of natural killer (NK) cells expressingthe cytotoxic receptor complex and/or a homing moiety as describedherein. In several embodiments, the engineered NK cells are autologouscells, while in some embodiments, the NK cells are allogeneic cells. Inseveral embodiments, NK cells are preferred because the naturalcytotoxic potential of NK cells is relatively high. In severalembodiments, it is unexpectedly beneficial that the engineered cellsdisclosed herein can further upregulate the cytotoxic activity of NKcells, leading to an even more effective activity against target cells(e.g., tumor or other diseased cells). Some embodiments of the methodsand compositions described herein relate to an NK that includes aCD19-directed chimeric receptor, or a nucleic acid encoding theCD19-directed chimeric receptor. Several embodiments of the methods andcompositions disclosed herein relate to NK cells engineered to express aCD19-directed chimeric receptor and a membrane-bound interleukin 15(mbIL15) co-stimulatory domain.

Hematopoietic Stem Cells for Cancer Immunotherapy

In some embodiments, hematopoietic stem cells (HSCs) are used in themethods of immunotherapy disclosed herein. In several embodiments, thecells are engineered to express a homing moiety and/or a cytotoxicreceptor complex. HSCs are used, in several embodiments, to leveragetheir ability to engraft for long-term blood cell production, whichcould result in a sustained source of targeted anti-cancer effectorcells, for example to combat cancer remissions. In several embodiments,this ongoing production helps to offset anergy or exhaustion of othercell types, for example due to the tumor microenvironment. In severalembodiments allogeneic HSCs are used, while in some embodiments,autologous HSCs are used. In several embodiments, HSCs are used incombination with one or more additional engineered cell type disclosedherein. Some embodiments of the methods and compositions describedherein relate to a stem cell, such as a hematopoietic stem cell, thatincludes a CD19-directed chimeric receptor, or a nucleic acid encodingthe CD19-directed chimeric receptor. Several embodiments of the methodsand compositions disclosed herein relate to stem cells, such ashematopoietic stem cells that are engineered to express a CD19-directedchimeric receptor and a membrane-bound interleukin 15 (mbIL15)co-stimulatory domain.

Extracellular domains (Tumor binder)

Some embodiments of the compositions and methods described herein relateto a chimeric receptor, such as a CD19-directed chimeric receptor, thatincludes an extracellular domain. In some embodiments, the extracellulardomain comprises a tumor-binding domain (also referred to as anantigen-binding protein or antigen-binding domain) as described herein.in some embodiments, the antigen-binding domain is derived from orcomprises wild-type or non-wild-type sequence of an antibody, anantibody fragment, an scFv, a Fv, a Fab, a (Fab′)2, a single domainantibody (SDAB), a vH or vL domain, a camelid VHH domain, or anon-immunoglobulin scaffold such as a DARPIN, an affibody, an affilin,an adnectin, an affitin, a repebody, a fynomer, an alphabody, an avimer,an atrimer, a centyrin, a pronectin, an anticalin, a kunitz domain, anArmadillo repeat protein, an autoantigen, a receptor or a ligand. Insome embodiments, the tumor-binding domain contains more than oneantigen binding domain. In embodiments, the antigen-binding domain isoperably linked directly or via an optional linker to the NH2-terminalend of a TCR domain (e.g. constant chains of TCR-alpha, TCR-beta1,TCR-beta2, preTCR-alpha, pre-TCR-alpha-De148, TCR-gamma, or TCR-delta)

Antigen-Binding Proteins

There are provided, in several embodiments, antigen-binding proteins. Asused herein, the term “antigen-binding protein” shall be given itsordinary meaning, and shall also refer to a protein comprising anantigen-binding fragment that binds to an antigen and, optionally, ascaffold or framework portion that allows the antigen-binding fragmentto adopt a conformation that promotes binding of the antigen-bindingprotein to the antigen. In some embodiments, the antigen is a cancerantigen (e.g., CD19) or a fragment thereof. In some embodiments, theantigen-binding fragment comprises at least one CDR from an antibodythat binds to the antigen. In some embodiments, the antigen-bindingfragment comprises all three CDRs from the heavy chain of an antibodythat binds to the antigen or from the light chain of an antibody thatbinds to the antigen. In still some embodiments, the antigen-bindingfragment comprises all six CDRs from an antibody that binds to theantigen (three from the heavy chain and three from the light chain). Inseveral embodiments, the antigen-binding fragment comprises one, two,three, four, five, or six CDRs from an antibody that binds to theantigen, and in several embodiments, the CDRs can be any combination ofheavy and/or light chain CDRs. The antigen-binding fragment in someembodiments is an antibody fragment.

Nonlimiting examples of antigen-binding proteins include antibodies,antibody fragments (e.g., an antigen-binding fragment of an antibody),antibody derivatives, and antibody analogs. Further specific examplesinclude, but are not limited to, a single-chain variable fragment(scFv), a nanobody (e.g. VH domain of camelid heavy chain antibodies;VHH fragment), a Fab fragment, a Fab′ fragment, a F(ab′)2 fragment, a Fvfragment, a Fd fragment, and a complementarity determining region (CDR)fragment. These molecules can be derived from any mammalian source, suchas human, mouse, rat, rabbit, or pig, dog, or camelid. Antibodyfragments may compete for binding of a target antigen with an intact(e.g., native) antibody and the fragments may be produced by themodification of intact antibodies (e.g. enzymatic or chemical cleavage)or synthesized de novo using recombinant DNA technologies or peptidesynthesis. The antigen-binding protein can comprise, for example, analternative protein scaffold or artificial scaffold with grafted CDRs orCDR derivatives. Such scaffolds include, but are not limited to,antibody-derived scaffolds comprising mutations introduced to, forexample, stabilize the three-dimensional structure of theantigen-binding protein as well as wholly synthetic scaffoldscomprising, for example, a biocompatible polymer. In addition, peptideantibody mimetics (“PAMs”) can be used, as well as scaffolds based onantibody mimetics utilizing fibronectin components as a scaffold.

In some embodiments, the antigen-binding protein comprises one or moreantibody fragments incorporated into a single polypeptide chain or intomultiple polypeptide chains. For instance, antigen-binding proteins caninclude, but are not limited to, a diabody; an intrabody; a domainantibody (single VL or VH domain or two or more VH domains joined by apeptide linker); a maxibody (2 scFvs fused to Fc region); a triabody; atetrabody; a minibody (scFv fused to CH3 domain); a peptibody (one ormore peptides attached to an Fc region); a linear antibody (a pair oftandem Fd segments (VH-CH1-VH-CH1) which, together with complementarylight chain polypeptides, form a pair of antigen binding regions); asmall modular immunopharmaceutical; and immunoglobulin fusion proteins(e.g. IgG-scFv, IgG-Fab, 2scFv-IgG, 4scFv-IgG, VH-IgG, IgG-VH, andFab-scFv-Fc).

In some embodiments, the antigen-binding protein has the structure of animmunoglobulin. As used herein, the term “immunoglobulin” shall be givenits ordinary meaning, and shall also refer to a tetrameric molecule,with each tetramer comprising two identical pairs of polypeptide chains,each pair having one “light” (about 25 kDa) and one “heavy” chain (about50-70 kDa). The amino-terminal portion of each chain includes a variableregion of about 100 to 110 or more amino acids primarily responsible forantigen recognition. The carboxy-terminal portion of each chain definesa constant region primarily responsible for effector function.

Within light and heavy chains, the variable (V) and constant regions (C)are joined by a “J” region of about 12 or more amino acids, with theheavy chain also including a “D” region of about 10 more amino acids.The variable regions of each light/heavy chain pair form the antibodybinding site such that an intact immunoglobulin has two binding sites.

Immunoglobulin chains exhibit the same general structure of relativelyconserved framework regions (FR) joined by three hypervariable regions,also called complementarity determining regions or CDRs. From N-terminusto C-terminus, both light and heavy chains comprise the domains FR1,CDR1, FR2, CDR2, FR3, CDR3 and FR4.

Human light chains are classified as kappa and lambda light chains. Anantibody “light chain”, refers to the smaller of the two types ofpolypeptide chains present in antibody molecules in their naturallyoccurring conformations. Kappa (K) and lambda (A) light chains refer tothe two major antibody light chain isotypes. A light chain may include apolypeptide comprising, from amino terminus to carboxyl terminus, asingle immunoglobulin light chain variable region (VL) and a singleimmunoglobulin light chain constant domain (CL).

Heavy chains are classified as mu (A delta (A), gamma (γ), alpha (a),and epsilon (E), and define the antibody's isotype as IgM, IgD, IgG,IgA, and IgE, respectively. An antibody “heavy chain” refers to thelarger of the two types of polypeptide chains present in antibodymolecules in their naturally occurring conformations, and which normallydetermines the class to which the antibody belongs. A heavy chain mayinclude a polypeptide comprising, from amino terminus to carboxylterminus, a single immunoglobulin heavy chain variable region (VH), animmunoglobulin heavy chain constant domain 1 (CH1), an immunoglobulinhinge region, an immunoglobulin heavy chain constant domain 2 (CH2), animmunoglobulin heavy chain constant domain 3 (CH3), and optionally animmunoglobulin heavy chain constant domain 4 (CH4).

The IgG-class is further divided into subclasses, namely, IgG1, IgG2,IgG3, and IgG4. The IgA-class is further divided into subclasses, namelyIgA1 and IgA2. The IgM has subclasses including, but not limited to,IgM1 and IgM2. The heavy chains in IgG, IgA, and IgD antibodies havethree domains (CH1, CH2, and CH3), whereas the heavy chains in IgM andIgE antibodies have four domains (CH1, CH2, CH3, and CH4). Theimmunoglobulin heavy chain constant domains can be from anyimmunoglobulin isotype, including subtypes. The antibody chains arelinked together via inter-polypeptide disulfide bonds between the CLdomain and the CH1 domain (e.g., between the light and heavy chain) andbetween the hinge regions of the antibody heavy chains.

In some embodiments, the antigen-binding protein is an antibody. Theterm “antibody”, as used herein, refers to a protein, or polypeptidesequence derived from an immunoglobulin molecule which specificallybinds with an antigen. Antibodies can be monoclonal, or polyclonal,multiple or single chain, or intact immunoglobulins, and may be derivedfrom natural sources or from recombinant sources. Antibodies can betetramers of immunoglobulin molecules. The antibody may be “humanized”,“chimeric” or non-human. An antibody may include an intactimmunoglobulin of any isotype, and includes, for instance, chimeric,humanized, human, and bispecific antibodies. An intact antibody willgenerally comprise at least two full-length heavy chains and twofull-length light chains. Antibody sequences can be derived solely froma single species, or can be “chimeric,” that is, different portions ofthe antibody can be derived from two different species as describedfurther below. Unless otherwise indicated, the term “antibody” alsoincludes antibodies comprising two substantially full-length heavychains and two substantially full-length light chains provided theantibodies retain the same or similar binding and/or function as theantibody comprised of two full length light and heavy chains. Forexample, antibodies having 1, 2, 3, 4, or 5 amino acid residuesubstitutions, insertions or deletions at the N-terminus and/orC-terminus of the heavy and/or light chains are included in thedefinition provided that the antibodies retain the same or similarbinding and/or function as the antibodies comprising two full lengthheavy chains and two full length light chains. Examples of antibodiesinclude monoclonal antibodies, polyclonal antibodies, chimericantibodies, humanized antibodies, human antibodies, bispecificantibodies, and synthetic antibodies. There is provided, in someembodiments, monoclonal and polyclonal antibodies. As used herein, theterm “polyclonal antibody” shall be given its ordinary meaning, andshall also refer to a population of antibodies that are typically widelyvaried in composition and binding specificity. As used herein, the term“monoclonal antibody” (“mAb”) shall be given its ordinary meaning, andshall also refer to one or more of a population of antibodies havingidentical sequences. Monoclonal antibodies bind to the antigen at aparticular epitope on the antigen.

In some embodiments, the antigen-binding protein is a fragment orantigen-binding fragment of an antibody. The term “antibody fragment”refers to at least one portion of an antibody, that retains the abilityto specifically interact with (e.g., by binding, steric hindrance,stabilizing/destabilizing, spatial distribution) an epitope of anantigen. Examples of antibody fragments include, but are not limited to,Fab, Fab′, F(ab′)2, Fv fragments, scFv antibody fragments,disulfide-linked Fvs (sdFv), a Fd fragment consisting of the VH and CHIdomains, linear antibodies, single domain antibodies such as sdAb(either vL or vH), camelid vHH domains, multi-specific antibodies formedfrom antibody fragments such as a bivalent fragment comprising two Fabfragments linked by a disulfide bridge at the hinge region, and anisolated CDR or other epitope binding fragments of an antibody. Anantigen binding fragment can also be incorporated into single domainantibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies,triabodies, tetrabodies, v-NAR and bis-scFv (see, e.g., Hollinger andHudson, Nature Biotechnology 23: 1126-1136, 2005). Antigen bindingfragments can also be grafted into scaffolds based on polypeptides suchas a fibronectin type III (Fn3)(see U.S. Pat. No. 6,703,199, whichdescribes fibronectin polypeptide mini bodies). An antibody fragment mayinclude a Fab, Fab′, F(ab′)2, and/or Fv fragment that contains at leastone CDR of an immunoglobulin that is sufficient to confer specificantigen binding to a cancer antigen (e.g., CD19). Antibody fragments maybe produced by recombinant DNA techniques or by enzymatic or chemicalcleavage of intact antibodies.

In some embodiments, Fab fragments are provided. A Fab fragment is amonovalent fragment having the VL, VH, CL and CH1 domains; a F(ab′)2fragment is a bivalent fragment having two Fab fragments linked by adisulfide bridge at the hinge region; a Fd fragment has the VH and CH1domains; an Fv fragment has the VL and VH domains of a single arm of anantibody; and a dAb fragment has a VH domain, a VL domain, or anantigen-binding fragment of a VH or VL domain. In some embodiments,these antibody fragments can be incorporated into single domainantibodies, single-chain antibodies, maxibodies, minibodies,intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv. Insome embodiments, the antibodies comprise at least one CDR as describedherein.

There is also provided for herein, in several embodiments, single-chainvariable fragments. As used herein, the term “single-chain variablefragment” (“scFv”) shall be given its ordinary meaning, and shall alsorefer to a fusion protein in which a VL and a VH region are joined via alinker (e.g., a synthetic sequence of amino acid residues) to form acontinuous protein chain wherein the linker is long enough to allow theprotein chain to fold back on itself and form a monovalent antigenbinding site). For the sake of clarity, unless otherwise indicated assuch, a “single-chain variable fragment” is not an antibody or anantibody fragment as defined herein. Diabodies are bivalent antibodiescomprising two polypeptide chains, wherein each polypeptide chaincomprises VH and VL domains joined by a linker that is configured toreduce or not allow for pairing between two domains on the same chain,thus allowing each domain to pair with a complementary domain on anotherpolypeptide chain. According to several embodiments, if the twopolypeptide chains of a diabody are identical, then a diabody resultingfrom their pairing will have two identical antigen binding sites.Polypeptide chains having different sequences can be used to make adiabody with two different antigen binding sites. Similarly, tribodiesand tetrabodies are antibodies comprising three and four polypeptidechains, respectively, and forming three and four antigen binding sites,respectively, which can be the same or different.

In several embodiments, the antigen-binding protein comprises one ormore CDRs. As used herein, the term “CDR” shall be given its ordinarymeaning, and shall also refer to the complementarity determining region(also termed “minimal recognition units” or “hypervariable region”)within antibody variable sequences. The CDRs permit the antigen-bindingprotein to specifically bind to a particular antigen of interest. Thereare three heavy chain variable region CDRs (CDRH1, CDRH2 and CDRH3) andthree light chain variable region CDRs (CDRL1, CDRL2 and CDRL3). TheCDRs in each of the two chains typically are aligned by the frameworkregions to form a structure that binds specifically to a specificepitope or domain on the target protein. From N-terminus to C-terminus,naturally-occurring light and heavy chain variable regions bothtypically conform to the following order of these elements: FR1, CDR1,FR2, CDR2, FR3, CDR3 and FR4. A numbering system has been devised forassigning numbers to amino acids that occupy positions in each of thesedomains. This numbering system is defined in Kabat Sequences of Proteinsof Immunological Interest (1987 and 1991, NIH, Bethesda, Md.), orChothia & Lesk, 1987, J. Mol. Biol. 196:901-917; Chothia et al., 1989,Nature 342:878-883. Complementarity determining regions (CDRs) andframework regions (FR) of a given antibody may be identified using thissystem. Other numbering systems for the amino acids in immunoglobulinchains include IMGT® (the international ImMunoGeneTics informationsystem; Lefranc et al, Dev. Comp. Immunol. 29:185-203; 2005) and AHo(Honegger and Pluckthun, J. Mol. Biol. 309(3):657-670; 2001). One ormore CDRs may be incorporated into a molecule either covalently ornoncovalently to make it an antigen-binding protein. In severalembodiments, the antigen-binding proteins provided herein comprise aheavy chain variable region selected from SEQ ID NO: 104 and SEQ ID NO:106. In several embodiments, the antigen-binding proteins providedherein comprise a light chain variable region selected from SEQ ID NO:105 and SEQ ID NO: 107.

In several embodiments, the antigen-binding protein has been modifiedfrom its original sequence, for example for purposes of improvingexpression, function, or reducing a potential immune response to theantigen-binding protein by a host. In several embodiments, theantigen-binding protein comprises a light chain variable region selectedfrom SEQ ID NO: 117, SEQ ID NO: 118, and SEQ ID NO: 119 and/or sequenceshaving at least 90% identity and/or homology (e.g., 90-95%, 95%, 96%,97%, 98%, 99%). In several embodiments, the light chain variable regiondiffers in sequence from SEQ ID NO: 117, SEQ ID NO: 118, or SEQ ID NO:119 by more than 5% (e.g., by 5-7%, 5-10%, 10-20% or higher) withligand-binding function (or other functionality) being similar,substantially similar or the same as SEQ ID NO: 117, SEQ ID NO: 118, orSEQ ID NO: 119. In several embodiments, the antigen-binding proteincomprises a heavy chain variable region selected from SEQ ID NO: 120,SEQ ID NO: 121, SEQ ID NO: 122, and SEQ ID NO: 123 and/or sequenceshaving at least 90% identity and/or homology (e.g., 90-95%, 95%, 96%,97%, 98%, 99%). In several embodiments, the heavy chain variable regiondiffers in sequence from SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122,or SEQ ID NO: 123 by more than 5% (e.g., by 5-7%, 5-10%, 10-20% orhigher) with ligand-binding function (or other functionality) beingsimilar, substantially similar or the same as SEQ ID NO: 120, SEQ ID NO:121, SEQ ID NO: 122, or SEQ ID NO: 123. Depending on the embodiment, anycombination of heavy and light chain regions may be used (e.g., inassembling a scFv). In several embodiments, the antigen-binding proteincomprises one or more CDRs selected from SEQ ID NO: 124, SEQ ID NO: 125,SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ IDNO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134,SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ IDNO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143,and SEQ ID NO: 144.

In additional embodiments, the CDRs are selected from SEQ ID NO: 108,SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ IDNO: 113, SEQ ID NO: 114, and SEQ ID NO: 115, in any combination. In oneembodiment, the CDRs are assembled to generate a CAR directed to CD19and comprising SEQ ID NO: 116.

In several embodiments, the antigen-binding protein comprises a heavychain having the sequence of SEQ ID NO: 88. In several embodiments, thatheavy chain is coupled with (e.g., as an scFv), one of the light chainsof SEQ ID NO: 89, SEQ ID NO: 90, and/or SEQ ID NO: 91. In severalembodiments, the antigen-binding protein comprises one of more CDRsselected from SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO:95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, and SEQID NO: 100.

In several embodiments, the antigen-binding protein comprises a lightchain region of an FMC63 antibody that has the sequence of SEQ ID NO:150. In several embodiments, the antigen-binding protein comprises alight chain region of an FMC63 antibody that has the sequence of SEQ IDNO: 148. In several embodiments, linkers are used between heavy andlight chains, and in some embodiments, the linker comprises the sequenceof SEQ ID NO: 149. In several embodiments, such heavy and light chainsare used in conjunction with a CD28 co-stimulatory domain, such as thatof SEQ ID NO: 153. Often a spacer is used to separate component parts ofa CAR. For example, in several embodiments, a spacer is used comprisingthe sequence of SEQ ID NO: 151. In several embodiments, a transmembranedomain having the sequence of SEQ ID NO: 152 is used. In severalembodiments, the CAR comprises a nucleic acid sequence that shares atleast about 90%, at least about 94%, at least about 95%, at least about96%, at least about 97%, at least about 98%, or at least about 99%,sequence identity, homology and/or functional equivalence with SEQ IDNO: 147.

In some embodiments, the antigen-binding proteins provided hereincomprise one or more CDR(s) as part of a larger polypeptide chain. Insome embodiments, the antigen-binding proteins covalently link the oneor more CDR(s) to another polypeptide chain. In some embodiments, theantigen-binding proteins incorporate the one or more CDR(s)noncovalently. In some embodiments, the antigen-binding proteins maycomprise at least one of the CDRs described herein incorporated into abiocompatible framework structure. In some embodiments, thebiocompatible framework structure comprises a polypeptide or portionthereof that is sufficient to form a conformationally stable structuralsupport, or framework, or scaffold, which is able to display one or moresequences of amino acids that bind to an antigen (e.g., CDRs, a variableregion, etc.) in a localized surface region. Such structures can be anaturally occurring polypeptide or polypeptide “fold” (a structuralmotif), or can have one or more modifications, such as additions,deletions and/or substitutions of amino acids, relative to a naturallyoccurring polypeptide or fold. Depending on the embodiment, thescaffolds can be derived from a polypeptide of a variety of differentspecies (or of more than one species), such as a human, a non-humanprimate or other mammal, other vertebrate, invertebrate, plant, bacteriaor virus.

Depending on the embodiment, the biocompatible framework structures arebased on protein scaffolds or skeletons other than immunoglobulindomains. In some such embodiments, those framework structures are basedon fibronectin, ankyrin, lipocalin, neocarzinostain, cytochrome b, CP1zinc finger, PST1, coiled coil, LACI-D1, Z domain and/or tendamistatdomains.

There is also provided, in some embodiments, antigen-binding proteinswith more than one binding site. In several embodiments, the bindingsites are identical to one another while in some embodiments the bindingsites are different from one another. For example, an antibody typicallyhas two identical binding sites, while a “bispecific” or “bifunctional”antibody has two different binding sites. The two binding sites of abispecific antigen-binding protein or antibody will bind to twodifferent epitopes, which can reside on the same or different proteintargets. In several embodiments, this is particularly advantageous, as abispecific chimeric antigen receptor can impart to an engineered cellthe ability to target multiple tumor markers. For example, CD19 and anadditional tumor marker, such as CD123, NKG2D or any other markerdisclosed herein or appreciated in the art as a tumor specific antigenor tumor associated antigen.

As used herein, the term “chimeric antibody” shall be given its ordinarymeaning, and shall also refer to an antibody that contains one or moreregions from one antibody and one or more regions from one or more otherantibodies. In some embodiments, one or more of the CDRs are derivedfrom an anti-cancer antigen (e.g., CD19) antibody. In severalembodiments, all of the CDRs are derived from an anti-cancer antigenantibody (such as an anti-CD19 antibody). In some embodiments, the CDRsfrom more than one anti-cancer antigen antibodies are mixed and matchedin a chimeric antibody. For instance, a chimeric antibody may comprise aCDR1 from the light chain of a first anti-cancer antigen antibody, aCDR2 and a CDR3 from the light chain of a second anti-cancer antigenantibody, and the CDRs from the heavy chain from a third anti-cancerantigen antibody. Further, the framework regions of antigen-bindingproteins disclosed herein may be derived from one of the sameanti-cancer antigen (e.g., CD19) antibodies, from one or more differentantibodies, such as a human antibody, or from a humanized antibody. Inone example of a chimeric antibody, a portion of the heavy and/or lightchain is identical with, homologous to, or derived from an antibody froma particular species or belonging to a particular antibody class orsubclass, while the remainder of the chain(s) is/are identical with,homologous to, or derived from an antibody or antibodies from anotherspecies or belonging to another antibody class or subclass. Alsoprovided herein are fragments of such antibodies that exhibit thedesired biological activity.

In some embodiments, an antigen-binding protein is provided comprising aheavy chain variable domain having at least 90% identity to the VHdomain amino acid sequence set forth in SEQ ID NO: 33. In someembodiments, the antigen-binding protein comprises a heavy chainvariable domain having at least 95% identity to the VH domain amino acidsequence set forth in SEQ ID NO: 33. In some embodiments, theantigen-binding protein comprises a heavy chain variable domain havingat least 96, 97, 98, or 99% identity to the VH domain amino acidsequence set forth in SEQ ID NO: 33. In several embodiments, the heavychain variable domain may have one or more additional mutations (e.g.,for purposes of humanization) in the VH domain amino acid sequence setforth in SEQ ID NO: 33, but retains specific binding to a cancer antigen(e.g., CD19). In several embodiments, the heavy chain variable domainmay have one or more additional mutations in the VH domain amino acidsequence set forth in SEQ ID NO: 33, but has improved specific bindingto a cancer antigen (e.g., CD19).

In some embodiments, the antigen-binding protein comprises a light chainvariable domain having at least 90% identity to the VL domain amino acidsequence set forth in SEQ ID NO: 32. In some embodiments, theantigen-binding protein comprises a light chain variable domain havingat least 95% identity to the VL domain amino acid sequence set forth inSEQ ID NO: 32. In some embodiments, the antigen-binding proteincomprises a light chain variable domain having at least 96, 97, 98, or99% identity to the VL domain amino acid sequence set forth in SEQ IDNO: 32. In several embodiments, the light chain variable domain may haveone or more additional mutations (e.g., for purposes of humanization) inthe VL domain amino acid sequence set forth in SEQ ID NO: 32, butretains specific binding to a cancer antigen (e.g., CD19). In severalembodiments, the light chain variable domain may have one or moreadditional mutations in the VL domain amino acid sequence set forth inSEQ ID NO: 32, but has improved specific binding to a cancer antigen(e.g., CD19).

In some embodiments, the antigen-binding protein comprises a heavy chainvariable domain having at least 90% identity to the VH domain amino acidsequence set forth in SEQ ID NO: 33, and a light chain variable domainhaving at least 90% identity to the VL domain amino acid sequence setforth in SEQ ID NO: 32. In some embodiments, the antigen-binding proteincomprises a heavy chain variable domain having at least 95% identity tothe VH domain amino acid sequence set forth in SEQ ID NO: 33, and alight chain variable domain having at least 95% identity to the VLdomain amino acid sequence set forth in SEQ ID NO: 32. In someembodiments, the antigen-binding protein comprises a heavy chainvariable domain having at least 96, 97, 98, or 99% identity to the VHdomain amino acid sequence set forth in SEQ ID NO: 33, and a light chainvariable domain having at least 96, 97, 98, or 99% identity to the VLdomain amino acid sequence set forth in SEQ ID NO: 32.

In some embodiments, the antigen-binding protein comprises a heavy chainvariable domain having the VH domain amino acid sequence set forth inSEQ ID NO: 33, and a light chain variable domain having the VL domainamino acid sequence set forth in SEQ ID NO: 32. In some embodiments, thelight-chain variable domain comprises a sequence of amino acids that isat least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or 100% identical to the sequence of a light chain variabledomain of SEQ ID NO: 32. In some embodiments, the light-chain variabledomain comprises a sequence of amino acids that is at least 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%identical to the sequence of a heavy chain variable domain in accordancewith SEQ ID NO: 33.

In some embodiments, the light chain variable domain comprises asequence of amino acids that is encoded by a nucleotide sequence that isat least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or 100% identical to the polynucleotide sequence SEQ ID NO: 32.In some embodiments, the light chain variable domain comprises asequence of amino acids that is encoded by a polynucleotide thathybridizes under moderately stringent conditions to the complement of apolynucleotide that encodes a light chain variable domain in accordancewith the sequence in SEQ ID NO: 32. In some embodiments, the light chainvariable domain comprises a sequence of amino acids that is encoded by apolynucleotide that hybridizes under stringent conditions to thecomplement of a polynucleotide that encodes a light chain variabledomain in accordance with the sequence in SEQ ID NO: 32.

In some embodiments, the heavy chain variable domain comprises asequence of amino acids that is at least 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequenceof a heavy chain variable domain in accordance with the sequence of SEQID NO: 33. In some embodiments, the heavy chain variable domaincomprises a sequence of amino acids that is encoded by a polynucleotidethat hybridizes under moderately stringent conditions to the complementof a polynucleotide that encodes a heavy chain variable domain inaccordance with the sequence of SEQ ID NO: 33. In some embodiments, theheavy chain variable domain comprises a sequence of amino acids that isencoded by a polynucleotide that hybridizes under stringent conditionsto the complement of a polynucleotide that encodes a heavy chainvariable domain in accordance with the sequence of SEQ ID NO: 33.

In several embodiments, additional anti-CD19 binding constructs areprovided. For example, in several embodiments, there is provided an scFvthat targets CD19 wherein the scFv comprises a heavy chain variableregion comprising the sequence of SEQ ID NO. 35. In some embodiments,the antigen-binding protein comprises a heavy chain variable domainhaving at least 95% identity to the HCV domain amino acid sequence setforth in SEQ ID NO: 35. In some embodiments, the antigen-binding proteincomprises a heavy chain variable domain having at least 96, 97, 98, or99% identity to the HCV domain amino acid sequence set forth in SEQ IDNO: 35. In several embodiments, the heavy chain variable domain may haveone or more additional mutations (e.g., for purposes of humanization) inthe HCV domain amino acid sequence set forth in SEQ ID NO: 35, butretains specific binding to a cancer antigen (e.g., CD19). In severalembodiments, the heavy chain variable domain may have one or moreadditional mutations in the HCV domain amino acid sequence set forth inSEQ ID NO: 35, but has improved specific binding to a cancer antigen(e.g., CD19).

Additionally, in several embodiments, an scFv that targets CD19comprises a light chain variable region comprising the sequence of SEQID NO. 36. In some embodiments, the antigen-binding protein comprises alight chain variable domain having at least 95% identity to the LCVdomain amino acid sequence set forth in SEQ ID NO: 36. In someembodiments, the antigen-binding protein comprises a light chainvariable domain having at least 96, 97, 98, or 99% identity to the LCVdomain amino acid sequence set forth in SEQ ID NO: 36. In severalembodiments, the light chain variable domain may have one or moreadditional mutations (e.g., for purposes of humanization) in the LCVdomain amino acid sequence set forth in SEQ ID NO: 36, but retainsspecific binding to a cancer antigen (e.g., CD19). In severalembodiments, the light chain variable domain may have one or moreadditional mutations in the LCV domain amino acid sequence set forth inSEQ ID NO: 36, but has improved specific binding to a cancer antigen(e.g., CD19).

In several embodiments, there is also provided an anti-CD19 bindingmoiety that comprises a light chain CDR comprising a first, second andthird complementarity determining region (LC CDR1, LC CDR2, and LC CDR3,respectively. In several embodiments, the anti-CD19 binding moietyfurther comprises a heavy chain CDR comprising a first, second and thirdcomplementarity determining region (HC CDR1, HC CDR2, and HC CDR3,respectively. In several embodiments, the LC CDR1 comprises the sequenceof SEQ ID NO. 37. In several embodiments, the LC CDR1 comprises an aminoacid sequence with at least about 85%, about 90%, about 95%, or about98% homology to the sequence of SEQ NO. 37. In several embodiments, theLC CDR2 comprises the sequence of SEQ ID NO. 38. In several embodiments,the LC CDR2 comprises an amino acid sequence with at least about 85%,about 90%, about 95%, or about 98% homology to the sequence of SEQ NO.38. In several embodiments, the LC CDR3 comprises the sequence of SEQ IDNO. 39. In several embodiments, the LC CDR3 comprises an amino acidsequence with at least about 85%, about 90%, about 95%, or about 98%homology to the sequence of SEQ NO. 39. In several embodiments, the HCCDR1 comprises the sequence of SEQ ID NO. 40. In several embodiments,the HC CDR1 comprises an amino acid sequence with at least about 85%,about 90%, about 95%, or about 98% homology to the sequence of SEQ NO.40. In several embodiments, the HC CDR2 comprises the sequence of SEQ IDNO. 41, 42, or 43. In several embodiments, the HC CDR2 comprises anamino acid sequence with at least about 85%, about 90%, about 95%, orabout 98% homology to the sequence of SEQ NO. 41, 42, or 43. In severalembodiments, the HC CDR3 comprises the sequence of SEQ ID NO. 44. Inseveral embodiments, the HC CDR3 comprises an amino acid sequence withat least about 85%, about 90%, about 95%, or about 98% homology to thesequence of SEQ NO. 44.

In several embodiments, there is also provided an anti-CD19 bindingmoiety that comprises a light chain variable region (VL) and a heavychain variable region (HL), the VL region comprising a first, second andthird complementarity determining region (VL CDR1, VL CDR2, and VL CDR3,respectively and the VH region comprising a first, second and thirdcomplementarity determining region (VH CDR1, VH CDR2, and VH CDR3,respectively. In several embodiments, the VL region comprises thesequence of SEQ ID NO. 45, 46, 47, or 48. In several embodiments, the VLregion comprises an amino acid sequence with at least about 85%, about90%, about 95%, or about 98% homology to the sequence of SEQ NO. 45, 46,47, or 48. In several embodiments, the VH region comprises the sequenceof SEQ ID NO. 49, 50, 51 or 52. In several embodiments, the VH regioncomprises an amino acid sequence with at least about 85%, about 90%,about 95%, or about 98% homology to the sequence of SEQ NO. 49, 50, 51or 52.

In several embodiments, there is also provided an anti-CD19 bindingmoiety that comprises a light chain CDR comprising a first, second andthird complementarity determining region (LC CDR1, LC CDR2, and LC CDR3,respectively. In several embodiments, the anti-CD19 binding moietyfurther comprises a heavy chain CDR comprising a first, second and thirdcomplementarity determining region (HC CDR1, HC CDR2, and HC CDR3,respectively. In several embodiments, the LC CDR1 comprises the sequenceof SEQ ID NO. 53. In several embodiments, the LC CDR1 comprises an aminoacid sequence with at least about 85%, about 90%, about 95%, or about98% homology to the sequence of SEQ NO. 53. In several embodiments, theLC CDR2 comprises the sequence of SEQ ID NO. 54. In several embodiments,the LC CDR2 comprises an amino acid sequence with at least about 85%,about 90%, about 95%, or about 98% homology to the sequence of SEQ NO.54. In several embodiments, the LC CDR3 comprises the sequence of SEQ IDNO. 55. In several embodiments, the LC CDR3 comprises an amino acidsequence with at least about 85%, about 90%, about 95%, or about 98%homology to the sequence of SEQ NO. 55. In several embodiments, the HCCDR1 comprises the sequence of SEQ ID NO. 56. In several embodiments,the HC CDR1 comprises an amino acid sequence with at least about 85%,about 90%, about 95%, or about 98% homology to the sequence of SEQ NO.56. In several embodiments, the HC CDR2 comprises the sequence of SEQ IDNO. 57. In several embodiments, the HC CDR2 comprises an amino acidsequence with at least about 85%, about 90%, about 95%, or about 98%homology to the sequence of SEQ NO. 57. In several embodiments, the HCCDR3 comprises the sequence of SEQ ID NO. 58. In several embodiments,the HC CDR3 comprises an amino acid sequence with at least about 85%,about 90%, about 95%, or about 98% homology to the sequence of SEQ NO.58.

Additional anti-CD19 binding moieties are known in the art, such asthose disclosed in, for example, U.S. Pat. No. 8,399,645, US PatentPublication No. 2018/0153977, US Patent Publication No. 2014/0271635, USPatent Publication No. 2018/0251514, and US Patent Publication No.2018/0312588, the entirety of each of which is incorporated by referenceherein.

Natural Killer Group Domains that Bind Tumor Ligands

In several embodiments, engineered immune cells such as NK cells areleveraged for their ability to recognize and destroy tumor cells. Forexample, an engineered NK cell may include a CD19-directed chimericreceptor or a nucleic acid encoding said chimeric receptor. NK cellsexpress both inhibitory and activating receptors on the cell surface.Inhibitory receptors bind self-molecules expressed on the surface ofhealthy cells (thus preventing immune responses against “self” cells),while the activating receptors bind ligands expressed on abnormal cells,such as tumor cells. When the balance between inhibitory and activatingreceptor activation is in favor of activating receptors, NK cellactivation occurs and target (e.g., tumor) cells are lysed.

Natural killer Group 2 member D (NKG2D) is an NK cell activatingreceptor that recognizes a variety of ligands expressed on cells. Thesurface expression of various NKG2D ligands is generally low in healthycells but is upregulated upon, for example, malignant transformation.Non-limiting examples of ligands recognized by NKG2D include, but arenot limited to, MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, andULBP6, as well as other molecules expressed on target cells that controlthe cytolytic or cytotoxic function of NK cells. In several embodiments,T cells are engineered to express an extracellular domain to binds toone or more tumor ligands and activate the T cell. For example, inseveral embodiments, T cells are engineered to express an NKG2D receptoras the binder/activation moiety. In several embodiments, engineeredcells as disclosed herein are engineered to express another member ofthe NKG2 family, e.g., NKG2A, NKG2C, and/or NKG2E. Combinations of suchreceptors are engineered in some embodiments. Moreover, in severalembodiments, other receptors are expressed, such as the Killer-cellimmunoglobulin-like receptors (KIRs).

In several embodiments, cells are engineered to express a cytotoxicreceptor complex comprising a full length NKG2D as an extracellularcomponent to recognize ligands on the surface of tumor cells (e.g.,liver cells). In one embodiment, full length NKG2D has the nucleic acidsequence of SEQ ID NO: 27. In several embodiments, the full lengthNKG2D, or functional fragment thereof is human NKG2D.

In several embodiments, cells are engineered to express a cytotoxicreceptor complex comprising a functional fragment of NKG2D as anextracellular component to recognize ligands on the surface of tumorcells or other diseased cells. In one embodiment, the functionalfragment of NKG2D has the nucleic acid sequence of SEQ ID NO: 25. Inseveral embodiments, the fragment of NKG2D is at least 70%, at least75%, at least 80%, at least 85%, at least 90%, or at least 95%homologous with full-length wild-type NKG2D. In several embodiments, thefragment may have one or more additional mutations from SEQ ID NO: 25,but retains, or in some embodiments, has enhanced, ligand-bindingfunction. In several embodiments, the functional fragment of NKG2Dcomprises the amino acid sequence of SEQ ID NO: 26. In severalembodiments, the NKG2D fragment is provided as a dimer, trimer, or otherconcatameric format, such embodiments providing enhanced ligand-bindingactivity. In several embodiments, the sequence encoding the NKG2Dfragment is optionally fully or partially codon optimized. In oneembodiment, a sequence encoding a codon optimized NKG2D fragmentcomprises the sequence of SEQ ID NO: 28.

Advantageously, according to several embodiments, the functionalfragment lacks its native transmembrane or intracellular domains butretains its ability to bind ligands of NKG2D as well as transduceactivation signals upon ligand binding. A further advantage of suchfragments is that expression of DAP10 to localize NKG2D to the cellmembrane is not required. Thus, in several embodiments, the cytotoxicreceptor complex encoded by the polypeptides disclosed herein does notcomprise DAP10. In several embodiments, immune cells, such as NK or Tcells, are engineered to express one or more chimeric receptors thattarget CD19 and an NGG2D ligand. Such cells, in several embodiments,also co-express mbIL15.

In several embodiments, the cytotoxic receptor complexes are configuredto dimerize. Dimerization may comprise homodimers or heterodimers,depending on the embodiment. In several embodiments, dimerizationresults in improved ligand recognition by the cytotoxic receptorcomplexes (and hence the NK cells expressing the receptor), resulting ina reduction in (or lack) of adverse toxic effects. In severalembodiments, the cytotoxic receptor complexes employ internal dimers, orrepeats of one or more component subunits. For example, in severalembodiments, the cytotoxic receptor complexes may optionally comprise afirst NKG2D extracellular domain coupled to a second NKG2D extracellulardomain, and a transmembrane/signaling region (or a separatetransmembrane region along with a separate signaling region).

In several embodiments, the various domains/subdomains are separated bya linker such as, a GS3 linker (SEQ ID NO: 15 and 16, nucleotide andprotein, respectively) is used (or a GSn linker). Other linkers usedaccording to various embodiments disclosed herein include, but are notlimited to those encoded by SEQ ID NO: 17, 19, 21 or 23. This providesthe potential to separate the various component parts of the receptorcomplex along the polynucleotide, which can enhance expression,stability, and/or functionality of the receptor complex.

Cytotoxic Signaling Complex

Some embodiments of the compositions and methods described herein relateto a chimeric receptor, such as a CD19-directed chimeric receptor, thatincludes a cytotoxic signaling complex. As disclosed herein, accordingto several embodiments, the provided cytotoxic receptor complexescomprise one or more transmembrane and/or intracellular domains thatinitiate cytotoxic signaling cascades upon the extracellular domain(s)binding to ligands on the surface of target cells. Certain embodimentsdisclosed herein relate to chimeric antigen receptor constructs whereinthe tumor-targeting domain (or CD19-directed domain) is coupled to acytotoxic signaling complex.

In several embodiments, the cytotoxic signaling complex comprises atleast one transmembrane domain, at least one co-stimulatory domain,and/or at least one signaling domain. In some embodiments, more than onecomponent part makes up a given domain—e.g., a co-stimulatory domain maycomprise two subdomains. Moreover, in some embodiments, a domain mayserve multiple functions, for example, a transmembrane domain may alsoserve to provide signaling function.

Transmembrane Domains

Some embodiments of the compositions and methods described herein relateto a chimeric receptor, such as a CD19-directed chimeric receptor, thatincludes a transmembrane domain. Some embodiments include atransmembrane domain from NKG2D or another transmembrane protein. Inseveral embodiments in which a transmembrane domain is employed, theportion of the transmembrane protein employed retains at least a portionof its normal transmembrane domain.

In several embodiments, however, the transmembrane domain comprises atleast a portion of CD8, a transmembrane glycoprotein normally expressedon both T cells and NK cells. In several embodiments, the transmembranedomain comprises CD8a. In several embodiments, the transmembrane domainis referred to as a “hinge”. In several embodiments, the “hinge” of CD8ahas the nucleic acid sequence of SEQ ID NO: 1. In several embodiments,the CD8a hinge is truncated or modified and is at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95% homologouswith the CD8a having the sequence of SEQ ID NO: 1. In severalembodiments, the “hinge” of CD8a comprises the amino acid sequence ofSEQ ID NO: 2. In several embodiments, the CD8a can be truncated ormodified, such that it is at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95% homologous with the sequence ofSEQ ID NO: 2.

In several embodiments, the transmembrane domain comprises a CD8atransmembrane region. In several embodiments, the CD8a transmembranedomain has the nucleic acid sequence of SEQ ID NO: 3. In severalembodiments, the CD8a hinge is truncated or modified and is at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least95% homologous with the CD8a having the sequence of SEQ ID NO: 3. Inseveral embodiments, the CD8a transmembrane domain comprises the aminoacid sequence of SEQ ID NO: 4. In several embodiments, the CD8a hinge istruncated or modified and is at least 70%, at least 75%, at least 80%,at least 85%, at least 90%, at least 95% homologous with the CD8a havingthe sequence of SEQ ID NO: 4.

Taken together in several embodiments, the CD8 hinge/transmembranecomplex is encoded by the nucleic acid sequence of SEQ ID NO: 13. Inseveral embodiments, the CD8 hinge/transmembrane complex is truncated ormodified and is at least 70%, at least 75%, at least 80%, at least 85%,at least 90%, at least 95% homologous with the CD8 hinge/transmembranecomplex having the sequence of SEQ ID NO: 13. In several embodiments,the CD8 hinge/transmembrane complex comprises the amino acid sequence ofSEQ ID NO: 14. In several embodiments, the CD8 hinge/transmembranecomplex hinge is truncated or modified and is at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95% homologouswith the CD8 hinge/transmembrane complex having the sequence of SEQ IDNO: 14.

In some embodiments, the transmembrane domain comprises a CD28transmembrane domain or a fragment thereof. In several embodiments, theCD28 transmembrane domain comprises the amino acid sequence of SEQ IDNO: 30. In several embodiments, the CD28 transmembrane domain complexhinge is truncated or modified and is at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95% homologous with theCD28 transmembrane domain having the sequence of SEQ ID NO: 30.

Co-Stimulatory Domains

Some embodiments of the compositions and methods described herein relateto a chimeric receptor, such as a CD19-directed chimeric receptor, thatincludes a co-stimulatory domain. In addition the various thetransmembrane domains and signaling domain (and the combinationtransmembrane/signaling domains), additional co-activating molecules canbe provided, in several embodiments. These can be certain moleculesthat, for example, further enhance activity of the immune cells.Cytokines may be used in some embodiments. For example, certaininterleukins, such as IL-2 and/or IL-15 as non-limiting examples, areused. In some embodiments, the immune cells for therapy are engineeredto express such molecules as a secreted form. In additional embodiments,such co-stimulatory domains are engineered to be membrane bound, actingas autocrine stimulatory molecules (or even as paracrine stimulators toneighboring cells delivered). In several embodiments, NK cells areengineered to express membrane-bound interleukin 15 (mbIL15). In suchembodiments, mbIL15 expression on the NK enhances the cytotoxic effectsof the engineered NK cell by enhancing the proliferation and/orlongevity of the NK cells. In several embodiments, mbIL15 has thenucleic acid sequence of SEQ ID NO: 11. In several embodiments, mbIL15can be truncated or modified, such that it is at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95% homologouswith the sequence of SEQ ID NO: 11. In several embodiments, the mbIL15comprises the amino acid sequence of SEQ ID NO: 12. In severalembodiments, the mbIL15 is truncated or modified and is at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%homologous with the mbIL15 having the sequence of SEQ ID NO: 12.

In some embodiments, the CD19-directed chimeric receptor or engineeredcytotoxic receptor complex is encoded by a polynucleotide that includesone or more cytosolic protease cleavage sites, for example a T2Acleavage site, a P2A cleavage site, an E2A cleavage site, and/or a F2Acleavage site. Such sites are recognized and cleaved by a cytosolicprotease, which can result in separation (and separate expression) ofthe various component parts of the receptor encoded by thepolynucleotide. As a result, depending on the embodiment, the variousconstituent parts of a CD19-directed chimeric receptor or engineeredcytotoxic receptor complex can be delivered to an NK cell or T cell in asingle vector or by multiple vectors. Thus, as shown schematically, inthe Figures, a construct can be encoded by a single polynucleotide, butalso include a cleavage site, such that downstream elements of theconstructs are expressed by the cells as a separate protein (as is thecase in some embodiments with IL-15). In several embodiments, a T2Acleavage site is used. In several embodiments, a T2A cleavage site hasthe nucleic acid sequence of SEQ ID NO: 9. In several embodiments, T2Acleavage site can be truncated or modified, such that it is at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least95% homologous with the sequence of SEQ ID NO: 9. In severalembodiments, the T2A cleavage site comprises the amino acid sequence ofSEQ ID NO: 10. In several embodiments, the T2A cleavage site istruncated or modified and is at least 70%, at least 75%, at least 80%,at least 85%, at least 90%, at least 95% homologous with the T2Acleavage site having the sequence of SEQ ID NO: 10.

Signaling Domains

Some embodiments of the compositions and methods described herein relateto a chimeric receptor, such as a CD19-directed chimeric receptor, thatincludes a signaling domain. For example, immune cells engineeredaccording to several embodiments disclosed herein may comprise at leastone subunit of the CD3 T cell receptor complex (or a fragment thereof).In several embodiments, the signaling domain comprises the CD3 zetasubunit. In several embodiments, the CD3 zeta is encoded by the nucleicacid sequence of SEQ ID NO: 7. In several embodiments, the CD3 zeta canbe truncated or modified, such that it is at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95% homologous with theCD3 zeta having the sequence of SEQ ID NO: 7. In several embodiments,the CD3 zeta domain comprises the amino acid sequence of SEQ ID NO: 8.In several embodiments, the CD3 zeta domain is truncated or modified andis at least 70%, at least 75%, at least 80%, at least 85%, at least 90%,at least 95% homologous with the CD3 zeta domain having the sequence ofSEQ ID NO: 8.

In several embodiments, unexpectedly enhanced signaling is achievedthrough the use of multiple signaling domains whose activities actsynergistically. For example, in several embodiments, the signalingdomain further comprises an OX40 domain. In several embodiments, theOX40 domain is an intracellular signaling domain. In severalembodiments, the OX40 intracellular signaling domain has the nucleicacid sequence of SEQ ID NO: 5. In several embodiments, the OX40intracellular signaling domain can be truncated or modified, such thatit is at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 95% homologous with the OX40 having the sequence of SEQ IDNO: 5. In several embodiments, the OX40 intracellular signaling domaincomprises the amino acid sequence of SEQ ID NO: 16. In severalembodiments, the OX40 intracellular signaling domain is truncated ormodified and is at least 70%, at least 75%, at least 80%, at least 85%,at least 90%, at least 95% homologous with the OX40 intracellularsignaling domain having the sequence of SEQ ID NO: 6. In severalembodiments, OX40 is used as the sole transmembrane/signaling domain inthe construct, however, in several embodiments, OX40 can be used withone or more other domains. For example, combinations of OX40 and CD3zetaare used in some embodiments. By way of further example, combinations ofCD28, OX40, 4-1 BB, and/or CD3zeta are used in some embodiments.

In several embodiments, the signaling domain comprises a 4-1BB domain.In several embodiments, the 4-1 BB domain is an intracellular signalingdomain. In several embodiments, the 4-1 BB intracellular signalingdomain comprises the amino acid sequence of SEQ ID NO: 29. In severalembodiments, the 4-1 BB intracellular signaling domain is truncated ormodified and is at least 70%, at least 75%, at least 80%, at least 85%,at least 90%, at least 95% homologous with the 4-1BB intracellularsignaling domain having the sequence of SEQ ID NO: 29. In severalembodiments, 4-1 BB is used as the sole transmembrane/signaling domainin the construct, however, in several embodiments, 4-1BB can be usedwith one or more other domains. For example, combinations of 4-1 BB andCD3zeta are used in some embodiments. By way of further example,combinations of CD28, OX40, 4-1 BB, and/or CD3zeta are used in someembodiments.

In several embodiments, the signaling domain comprises a CD28 domain. Inseveral embodiments the CD28 domain is an intracellular signalingdomain. In several embodiments, the CD28 intracellular signaling domaincomprises the amino acid sequence of SEQ ID NO: 31. In severalembodiments, the CD28 intracellular signaling domain is truncated ormodified and is at least 70%, at least 75%, at least 80%, at least 85%,at least 90%, at least 95% homologous with the CD28 intracellularsignaling domain having the sequence of SEQ ID NO: 31. In severalembodiments, CD28 is used as the sole transmembrane/signaling domain inthe construct, however, in several embodiments, CD28 can be used withone or more other domains. For example, combinations of CD28 and CD3zetaare used in some embodiments. By way of further example, combinations ofCD28, OX40, 4-1 BB, and/or CD3zeta are used in some embodiments.

Cytotoxic Receptor Complex Constructs

Some embodiments of the compositions and methods described herein relateto a chimeric receptor, such as a CD19-directed chimeric receptor, thatcomprises a cytotoxic receptor complex or cytotoxic receptor complexconstruct. In line with the above, a variety of cytotoxic receptorcomplexes (also referred to as cytotoxic receptors) are provided forherein. The expression of these complexes in immune cells, such as Tcells and/or NK cells, allows the targeting and destruction ofparticular target cells, such as cancerous cells. Non-limiting examplesof such cytotoxic receptor complexes are discussed in more detail below.

Chimeric Antigen Receptor Cytotoxic Receptor Complex Constructs

In several embodiments, there are provided for herein a variety ofcytotoxic receptor complexes (also referred to as cytotoxic receptors)are provided for herein with the general structure of a chimeric antigenreceptor. FIGS. 1A, 1B, and 2 schematically depict non-limitingschematics of constructs that include an anti-CD19 moiety that binds totumor antigens or tumor-associated antigens expressed on the surface ofcancer cells and activates the engineered cell expressing the chimericantigen receptor. As shown in the figures, several embodiments of thechimeric receptor include an anti-CD19 moiety, a CD8a hinge domain, anIg4 SH domain (or hinge), a CD8a transmembrane domain, a CD28transmembrane domain, an OX40 domain, a 4-1BB domain, a CD28 domain, aCD3 ITAM domain or subdomain, a CD3zeta domain, an NKp80 domain, a CD16IC domain, a 2A cleavage site, and a membrane-bound IL-15 domain(though, as above, in several embodiments soluble IL-15 is used). Inseveral embodiments, the binding and activation functions are engineeredto be performed by separate domains. Several embodiments relate tocomplexes with more than one anti-CD19 moiety or other binder/activationmoiety. In some embodiments, the binder/activation moiety targets othermarkers besides CD19, such as a cancer target described herein. Inseveral embodiments, the general structure of the chimeric antigenreceptor construct includes a hinge and/or transmembrane domain. Thesemay, in some embodiments, be fulfilled by a single domain, or aplurality of subdomains may be used, in several embodiments. Thereceptor complex further comprises a signaling domain, which transducessignals after binding of the homing moiety to the target cell,ultimately leading to the cytotoxic effects on the target cell. Inseveral embodiments, the complex further comprises a co-stimulatorydomain, which operates, synergistically, in several embodiments, toenhance the function of the signaling domain. Expression of thesecomplexes in immune cells, such as T cells and/or NK cells, allows thetargeting and destruction of particular target cells, such as cancerouscells that express CD19. Some such receptor complexes comprise anextracellular domain comprising an anti-CD19 moiety, or CD19-bindingmoiety, that binds CD19 on the surface of target cells and activates theengineered cell. The CD3zeta ITAM subdomain may act in concert as asignaling domain. The IL-15 domain, e.g., mbIL-15 domain, may acting asa co-stimulatory domain. The IL-15 domain, e.g. mbIL-15 domain, mayrender immune cells (e.g., NK or T cells) expressing it particularlyefficacious against target tumor cells. It shall be appreciated that theIL-15 domain, such as an mbIL-15 domain, can, in accordance with severalembodiments, be encoded on a separate construct. Additionally, each ofthe components may be encoded in one or more separate constructs. Insome embodiments, the cytotoxic receptor or CD19-directed receptorcomprises a sequence of amino acids that is at least 70%, 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, or a rangedefined by any two of the aforementioned percentages, identical to thesequence of SEQ ID NO: 34.

In one embodiment, there is provided a polynucleotide encoding ananti-CD19moiety/CD8hinge-CD8TM/OX40/CD3zeta chimeric antigen receptorcomplex (see FIG. 1A, CD19-1a). The polynucleotide comprises or iscomposed of an anti-CD19 moiety, a CD8a hinge, a CD8a transmembranedomain, an OX40 domain, and a CD3zeta domain as described herein. Inseveral embodiments, this receptor complex is encoded by a nucleic acidmolecule comprising a sequence obtained from a combination of sequencesdisclosed herein, or comprises an amino acid sequence obtained from acombination of sequences disclosed herein. In several embodiments, theencoding nucleic acid sequence, or the amino acid sequence, comprises asequence in accordance with one or more SEQ ID NOS as described herein,such as those included herein as examples of constituent parts. Inseveral embodiments, the encoding nucleic acid sequence, or the aminoacid sequence, comprises a sequence that shares at least about 90%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99%, sequence identity,homology and/or functional equivalence with a sequence resulting fromthe combination one or more SEQ ID NOS as described herein.

In several embodiments, there is provided a polynucleotide encoding ananti-CD19moiety/CD8hinge-CD8TM/OX40/CD3zeta/2A/mIL-15 chimeric antigenreceptor complex (see FIG. 1A, CD19-1b). The polynucleotide comprises oris composed of an anti-CD19 moiety, a CD8a hinge, a CD8a transmembranedomain, an OX40 domain, a CD3zeta domain, a 2A cleavage site, and anmIL-15 domain as described herein. In several embodiments, this receptorcomplex is encoded by a nucleic acid molecule comprising a sequenceobtained from a combination of sequences disclosed herein, or comprisesan amino acid sequence obtained from a combination of sequencesdisclosed herein. In several embodiments, the encoding nucleic acidsequence, or the amino acid sequence, comprises a sequence in accordancewith one or more SEQ ID NOS as described herein, such as those includedherein as examples of constituent parts. In several embodiments, theencoding nucleic acid sequence, or the amino acid sequence, comprises asequence that shares at least about 90%, at least about 94%, at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,or at least about 99%, sequence identity, homology and/or functionalequivalence with a sequence resulting from the combination one or moreSEQ ID NOS as described herein.

In several embodiments, there is provided a polynucleotide encoding ananti-CD19moiety/Ig4SH-CD8TM/4-1BB/CD3zeta chimeric antigen receptorcomplex (see FIG. 1A, CD19-2a). The polynucleotide comprises or iscomposed of an anti-CD19 moiety, a Ig4 SH domain, a CD8a transmembranedomain, a 4-1BB domain, and a CD3zeta domain as described herein. Inseveral embodiments, this receptor complex is encoded by a nucleic acidmolecule comprising a sequence obtained from a combination of sequencesdisclosed herein, or comprises an amino acid sequence obtained from acombination of sequences disclosed herein. In several embodiments, theencoding nucleic acid sequence, or the amino acid sequence, comprises asequence in accordance with one or more SEQ ID NOS as described herein,such as those included herein as examples of constituent parts. Inseveral embodiments, the encoding nucleic acid sequence, or the aminoacid sequence, comprises a sequence that shares at least about 90%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99%, sequence identity,homology and/or functional equivalence with a sequence resulting fromthe combination one or more SEQ ID NOS as described herein.

In several embodiments, there is provided a polynucleotide encoding ananti-CD19moiety/Ig4SH-CD8TM/4-1BB/CD3zeta/2A/mIL-15 chimeric antigenreceptor complex (see FIG. 1A, CD19-2b).

The polynucleotide comprises or is composed of an anti-CD19 moiety, aIg4 SH domain, a CD8a transmembrane domain, a 4-1 BB domain, a CD3zetadomain, a 2A cleavage site, and an mIL-15 domain as described herein. Inseveral embodiments, this receptor complex is encoded by a nucleic acidmolecule comprising a sequence obtained from a combination of sequencesdisclosed herein, or comprises an amino acid sequence obtained from acombination of sequences disclosed herein. In several embodiments, theencoding nucleic acid sequence, or the amino acid sequence, comprises asequence in accordance with one or more SEQ ID NOS as described herein,such as those included herein as examples of constituent parts. Inseveral embodiments, the encoding nucleic acid sequence, or the aminoacid sequence, comprises a sequence that shares at least about 90%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99%, sequence identity,homology and/or functional equivalence with a sequence resulting fromthe combination one or more SEQ ID NOS as described herein.

In several embodiments, there is provided a polynucleotide encoding ananti-CD19moiety/CD8hinge-CD28TM/CD28/CD3zeta chimeric antigen receptorcomplex (see FIG. 1A, CD19-3a). The polynucleotide comprises or iscomposed of an anti-CD19 moiety, a CD8a hinge, a CD28 transmembranedomain, a CD28 domain, and a CD3zeta domain as described herein. Inseveral embodiments, this receptor complex is encoded by a nucleic acidmolecule comprising a sequence obtained from a combination of sequencesdisclosed herein, or comprises an amino acid sequence obtained from acombination of sequences disclosed herein. In several embodiments, theencoding nucleic acid sequence, or the amino acid sequence, comprises asequence in accordance with one or more SEQ ID NOS as described herein,such as those included herein as examples of constituent parts. Inseveral embodiments, the encoding nucleic acid sequence, or the aminoacid sequence, comprises a sequence that shares at least about 90%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99%, sequence identity,homology and/or functional equivalence with a sequence resulting fromthe combination one or more SEQ ID NOS as described herein.

In several embodiments, there is provided a polynucleotide encoding ananti-CD19moiety/CD8hinge-CD28TM/CD28/CD3zeta/2A/mIL-15 chimeric antigenreceptor complex (see FIG. 1A, CD19-3b). The polynucleotide comprises oris composed of an anti-CD19 moiety, a CD8a hinge, a CD28 transmembranedomain, a CD28 domain, a CD3zeta domain, a 2A cleavage site, and anmIL-15 domain as described herein. In several embodiments, this receptorcomplex is encoded by a nucleic acid molecule comprising a sequenceobtained from a combination of sequences disclosed herein, or comprisesan amino acid sequence obtained from a combination of sequencesdisclosed herein. In several embodiments, the encoding nucleic acidsequence, or the amino acid sequence, comprises a sequence in accordancewith one or more SEQ ID NOS as described herein, such as those includedherein as examples of constituent parts. In several embodiments, theencoding nucleic acid sequence, or the amino acid sequence, comprises asequence that shares at least about 90%, at least about 94%, at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,or at least about 99%, sequence identity, homology and/or functionalequivalence with a sequence resulting from the combination one or moreSEQ ID NOS as described herein.

In several embodiments, there is provided a polynucleotide encoding ananti-CD19moiety/Ig4SH-CD28TM/CD28/CD3zeta chimeric antigen receptorcomplex (see FIG. 1A, CD19-4a). The polynucleotide comprises or iscomposed of an anti-CD19 moiety, an Ig4 SH domain, a CD28 transmembranedomain, a CD28 domain, and a CD3zeta domain as described herein. Inseveral embodiments, this receptor complex is encoded by a nucleic acidmolecule comprising a sequence obtained from a combination of sequencesdisclosed herein, or comprises an amino acid sequence obtained from acombination of sequences disclosed herein. In several embodiments, theencoding nucleic acid sequence, or the amino acid sequence, comprises asequence in accordance with one or more SEQ ID NOS as described herein,such as those included herein as examples of constituent parts. Inseveral embodiments, the encoding nucleic acid sequence, or the aminoacid sequence, comprises a sequence that shares at least about 90%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99%, sequence identity,homology and/or functional equivalence with a sequence resulting fromthe combination one or more SEQ ID NOS as described herein.

In several embodiments, there is provided a polynucleotide encoding ananti-CD19moiety/Ig4SH-CD28TM/CD28/CD3zeta/2A/mIL-15 chimeric antigenreceptor complex (see FIG. 1A, CD19-4b). The polynucleotide comprises oris composed of an anti-CD19 moiety, an Ig4 SH domain, a CD28transmembrane domain, a CD28 domain, a CD3zeta domain, a 2A cleavagesite, and an mIL-15 domain as described herein. In several embodiments,this receptor complex is encoded by a nucleic acid molecule comprising asequence obtained from a combination of sequences disclosed herein, orcomprises an amino acid sequence obtained from a combination ofsequences disclosed herein. In several embodiments, the encoding nucleicacid sequence, or the amino acid sequence, comprises a sequence inaccordance with one or more SEQ ID NOS as described herein, such asthose included herein as examples of constituent parts. In severalembodiments, the encoding nucleic acid sequence, or the amino acidsequence, comprises a sequence that shares at least about 90%, at leastabout 94%, at least about 95%, at least about 96%, at least about 97%,at least about 98%, or at least about 99%, sequence identity, homologyand/or functional equivalence with a sequence resulting from thecombination one or more SEQ ID NOS as described herein.

In several embodiments, there is provided a polynucleotide encoding ananti-CD19moiety/Ig4SH-CD8TM/OX40/CD3zeta chimeric antigen receptorcomplex (see FIG. 2A, CD19-5a). The polynucleotide comprises or iscomposed of an anti-CD19 moiety, a Ig4 SH domain, a CD8a transmembranedomain, an OX40 domain, and a CD3zeta domain as described herein. Inseveral embodiments, this receptor complex is encoded by a nucleic acidmolecule comprising a sequence obtained from a combination of sequencesdisclosed herein, or comprises an amino acid sequence obtained from acombination of sequences disclosed herein. In several embodiments, theencoding nucleic acid sequence, or the amino acid sequence, comprises asequence in accordance with one or more SEQ ID NOS as described herein,such as those included herein as examples of constituent parts. Inseveral embodiments, the encoding nucleic acid sequence, or the aminoacid sequence, comprises a sequence that shares at least about 90%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99%, sequence identity,homology and/or functional equivalence with a sequence resulting fromthe combination one or more SEQ ID NOS as described herein.

In several embodiments, there is provided a polynucleotide encoding ananti-CD19moiety/Ig4SH-CD8TM/OX40/CD3zeta/2A/mIL-15 chimeric antigenreceptor complex (see FIG. 2A, CD19-5b). The polynucleotide comprises oris composed of an anti-CD19 moiety, a Ig4 SH domain, a CD8atransmembrane domain, an OX40 domain, a CD3zeta domain, a 2A cleavagesite, and an mIL-15 domain as described herein. In several embodiments,this receptor complex is encoded by a nucleic acid molecule comprising asequence obtained from a combination of sequences disclosed herein, orcomprises an amino acid sequence obtained from a combination ofsequences disclosed herein. In several embodiments, the encoding nucleicacid sequence, or the amino acid sequence, comprises a sequence inaccordance with one or more SEQ ID NOS as described herein, such asthose included herein as examples of constituent parts. In severalembodiments, the encoding nucleic acid sequence, or the amino acidsequence, comprises a sequence that shares at least about 90%, at leastabout 94%, at least about 95%, at least about 96%, at least about 97%,at least about 98%, or at least about 99%, sequence identity, homologyand/or functional equivalence with a sequence resulting from thecombination one or more SEQ ID NOS as described herein.

In several embodiments, there is provided a polynucleotide encoding ananti-CD19moiety/CD8hinge-CD3aTM/CD28/CD3zeta chimeric antigen receptorcomplex (see FIG. 1B, CD19-6a). The polynucleotide comprises or iscomposed of an anti-CD19 moiety, a CD8a hinge, a CD3a transmembranedomain, a CD28 domain, and a CD3zeta domain as described herein. Inseveral embodiments, this receptor complex is encoded by a nucleic acidmolecule comprising a sequence obtained from a combination of sequencesdisclosed herein, or comprises an amino acid sequence obtained from acombination of sequences disclosed herein. In several embodiments, theencoding nucleic acid sequence, or the amino acid sequence, comprises asequence in accordance with one or more SEQ ID NOS as described herein,such as those included herein as examples of constituent parts. Inseveral embodiments, the encoding nucleic acid sequence, or the aminoacid sequence, comprises a sequence that shares at least about 90%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99%, sequence identity,homology and/or functional equivalence with a sequence resulting fromthe combination one or more SEQ ID NOS as described herein.

In several embodiments, there is provided a polynucleotide encoding ananti-CD19moiety/CD8hinge-CD3aTM/CD28/CD3zeta/2A/mIL-15 chimeric antigenreceptor complex (see FIG. 1B, CD19-6b). The polynucleotide comprises oris composed of an anti-CD19 moiety, a CD8a hinge, a CD3a transmembranedomain, a CD28 domain, a CD3zeta domain, a 2A cleavage site, and anmIL-15 domain as described herein. In several embodiments, this receptorcomplex is encoded by a nucleic acid molecule comprising a sequenceobtained from a combination of sequences disclosed herein, or comprisesan amino acid sequence obtained from a combination of sequencesdisclosed herein. In several embodiments, the encoding nucleic acidsequence, or the amino acid sequence, comprises a sequence in accordancewith one or more SEQ ID NOS as described herein, such as those includedherein as examples of constituent parts. In several embodiments, theencoding nucleic acid sequence, or the amino acid sequence, comprises asequence that shares at least about 90%, at least about 94%, at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,or at least about 99%, sequence identity, homology and/or functionalequivalence with a sequence resulting from the combination one or moreSEQ ID NOS as described herein.

In several embodiments, there is provided a polynucleotide encoding ananti-CD19moiety/CD8hinge-CD28TM/CD28/4-1BB/CD3zeta chimeric antigenreceptor complex (see FIG. 1B, CD19-7a). The polynucleotide comprises oris composed of an anti-CD19 moiety, a CD8a hinge, a CD28 transmembranedomain, a CD28 domain, a 4-1 BB domain, and a CD3zeta domain asdescribed herein. In several embodiments, this receptor complex isencoded by a nucleic acid molecule comprising a sequence obtained from acombination of sequences disclosed herein, or comprises an amino acidsequence obtained from a combination of sequences disclosed herein. Inseveral embodiments, the encoding nucleic acid sequence, or the aminoacid sequence, comprises a sequence in accordance with one or more SEQID NOS as described herein, such as those included herein as examples ofconstituent parts. In several embodiments, the encoding nucleic acidsequence, or the amino acid sequence, comprises a sequence that sharesat least about 90%, at least about 94%, at least about 95%, at leastabout 96%, at least about 97%, at least about 98%, or at least about99%, sequence identity, homology and/or functional equivalence with asequence resulting from the combination one or more SEQ ID NOS asdescribed herein.

In several embodiments, there is provided a polynucleotide encoding ananti-CD19moiety/CD8hinge-CD28TM/CD28/4-1BB/CD3zeta/2A/mIL-15 chimericantigen receptor complex (see FIG. 1B, CD19-7b). The polynucleotidecomprises or is composed of an anti-CD19 moiety, a CD8a hinge, a CD28transmembrane domain, a CD28 domain, a 4-1BB domain, a CD3zeta domain, a2A cleavage site, and an mIL-15 domain as described herein. In severalembodiments, this receptor complex is encoded by a nucleic acid moleculecomprising a sequence obtained from a combination of sequences disclosedherein, or comprises an amino acid sequence obtained from a combinationof sequences disclosed herein. In several embodiments, the encodingnucleic acid sequence, or the amino acid sequence, comprises a sequencein accordance with one or more SEQ ID NOS as described herein, such asthose included herein as examples of constituent parts. In severalembodiments, the encoding nucleic acid sequence, or the amino acidsequence, comprises a sequence that shares at least about 90%, at leastabout 94%, at least about 95%, at least about 96%, at least about 97%,at least about 98%, or at least about 99%, sequence identity, homologyand/or functional equivalence with a sequence resulting from thecombination one or more SEQ ID NOS as described herein.

In several embodiments, there is provided a polynucleotide encoding ananti-CD19moiety/CD8 alpha hinge/CD8 alpha TM/4-1BB/CD3zeta chimericantigen receptor complex (see FIG. 2, CD19-8a). The polynucleotidecomprises or is composed of an anti-CD19 moiety, a CD8a hinge, a CD8atransmembrane domain, a 4-1 BB domain, and a CD3zeta domain as describedherein. In several embodiments, this receptor complex is encoded by anucleic acid molecule comprising a sequence obtained from a combinationof sequences disclosed herein, or comprises an amino acid sequenceobtained from a combination of sequences disclosed herein. In severalembodiments, the encoding nucleic acid sequence, or the amino acidsequence, comprises a sequence in accordance with one or more SEQ ID NOSas described herein, such as those included herein as examples ofconstituent parts. In several embodiments, the encoding nucleic acidsequence, or the amino acid sequence, comprises a sequence that sharesat least about 90%, at least about 94%, at least about 95%, at leastabout 96%, at least about 97%, at least about 98%, or at least about99%, sequence identity, homology and/or functional equivalence with asequence resulting from the combination one or more SEQ ID NOS asdescribed herein.

In several embodiments, there is provided a polynucleotide encoding ananti-CD19moiety/CD8 alpha hinge/CD8 alpha TM/4-1BB/CD3zeta/2A/mIL-15chimeric antigen receptor complex (see FIG. 2, CD19-8b). Thepolynucleotide comprises or is composed of an anti-CD19 moiety, a CD8ahinge, a CD8a transmembrane domain, a 4-1 BB domain, a CD3zeta domain, a2A cleavage site, and an mIL-15 domain as described herein. In severalembodiments, this receptor complex is encoded by a nucleic acid moleculecomprising a sequence obtained from a combination of sequences disclosedherein, or comprises an amino acid sequence obtained from a combinationof sequences disclosed herein. In several embodiments, the encodingnucleic acid sequence, or the amino acid sequence, comprises a sequencein accordance with one or more SEQ ID NOS as described herein, such asthose included herein as examples of constituent parts. In severalembodiments, the encoding nucleic acid sequence, or the amino acidsequence, comprises a sequence that shares at least about 90%, at leastabout 94%, at least about 95%, at least about 96%, at least about 97%,at least about 98%, or at least about 99%, sequence identity, homologyand/or functional equivalence with a sequence resulting from thecombination one or more SEQ ID NOS as described herein.

In several embodiments, there is provided a polynucleotide encoding ananti-CD19moiety/CD8 alpha hinge/CD3 TM/4-1 BB/CD3zeta chimeric antigenreceptor complex (see FIG. 2, CD19-39_5a). The polynucleotide comprisesor is composed of an anti-CD19 moiety, a CD8a hinge, a CD3 transmembranedomain, a 4-1BB domain, and a CD3zeta domain as described herein. Inseveral embodiments, this receptor complex is encoded by a nucleic acidmolecule comprising a sequence obtained from a combination of sequencesdisclosed herein, or comprises an amino acid sequence obtained from acombination of sequences disclosed herein. In several embodiments, theencoding nucleic acid sequence, or the amino acid sequence, comprises asequence in accordance with one or more SEQ ID NOS as described herein,such as those included herein as examples of constituent parts. Inseveral embodiments, the encoding nucleic acid sequence, or the aminoacid sequence, comprises a sequence that shares at least about 90%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99%, sequence identity,homology and/or functional equivalence with a sequence resulting fromthe combination one or more SEQ ID NOS as described herein.

In several embodiments, there is provided a polynucleotide encoding ananti-CD19moiety/CD8 alpha hinge/CD3 TM/4-1BB/CD3zeta/2A/mIL-15 chimericantigen receptor complex (see FIG. 2, CD19-39_5b). The polynucleotidecomprises or is composed of an anti-CD19 moiety, a CD8a hinge, a CD8atransmembrane domain, a 4-1 BB domain, a CD3zeta domain, a 2A cleavagesite, and an mIL-15 domain as described herein. In several embodiments,this receptor complex is encoded by a nucleic acid molecule comprising asequence obtained from a combination of sequences disclosed herein, orcomprises an amino acid sequence obtained from a combination ofsequences disclosed herein. In several embodiments, the encoding nucleicacid sequence, or the amino acid sequence, comprises a sequence inaccordance with one or more SEQ ID NOS as described herein, such asthose included herein as examples of constituent parts. In severalembodiments, the encoding nucleic acid sequence, or the amino acidsequence, comprises a sequence that shares at least about 90%, at leastabout 94%, at least about 95%, at least about 96%, at least about 97%,at least about 98%, or at least about 99%, sequence identity, homologyand/or functional equivalence with a sequence resulting from thecombination one or more SEQ ID NOS as described herein.

In several embodiments, there is provided a polynucleotide encoding ananti-CD19moiety/CD8 alpha hinge/CD3 TM/4-1BB/NKp80 chimeric antigenreceptor complex (see FIG. 2, CD19-39_6a). The polynucleotide comprisesor is composed of an anti-CD19 moiety, a CD8a hinge, a CD3 transmembranedomain, a 4-1BB domain, and an NKp80 domain as described herein. Inseveral embodiments, this receptor complex is encoded by a nucleic acidmolecule comprising a sequence obtained from a combination of sequencesdisclosed herein, or comprises an amino acid sequence obtained from acombination of sequences disclosed herein. In several embodiments, theencoding nucleic acid sequence, or the amino acid sequence, comprises asequence in accordance with one or more SEQ ID NOS as described herein,such as those included herein as examples of constituent parts. Inseveral embodiments, the encoding nucleic acid sequence, or the aminoacid sequence, comprises a sequence that shares at least about 90%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99%, sequence identity,homology and/or functional equivalence with a sequence resulting fromthe combination one or more SEQ ID NOS as described herein.

In several embodiments, there is provided a polynucleotide encoding ananti-CD19moiety/CD8 alpha hinge/CD3 TM/4-1BB/NKp80/2A/mIL-15 chimericantigen receptor complex (see FIG. 2, CD19-39_6b). The polynucleotidecomprises or is composed of an anti-CD19 moiety, a CD8a hinge, a CD8atransmembrane domain, a 4-1 BB domain, an NKp80 domain, a 2A cleavagesite, and an mIL-15 domain as described herein. In several embodiments,this receptor complex is encoded by a nucleic acid molecule comprising asequence obtained from a combination of sequences disclosed herein, orcomprises an amino acid sequence obtained from a combination ofsequences disclosed herein. In several embodiments, the encoding nucleicacid sequence, or the amino acid sequence, comprises a sequence inaccordance with one or more SEQ ID NOS as described herein, such asthose included herein as examples of constituent parts. In severalembodiments, the encoding nucleic acid sequence, or the amino acidsequence, comprises a sequence that shares at least about 90%, at leastabout 94%, at least about 95%, at least about 96%, at least about 97%,at least about 98%, or at least about 99%, sequence identity, homologyand/or functional equivalence with a sequence resulting from thecombination one or more SEQ ID NOS as described herein.

In several embodiments, there is provided a polynucleotide encoding ananti-CD19moiety/CD8 alpha hinge/CD3 TM/CD16 intracellular domain/4-1BBchimeric antigen receptor complex (see FIG. 2, CD19-39_10a). Thepolynucleotide comprises or is composed of an anti-CD19 moiety, a CD8ahinge, a CD3 transmembrane domain, CD16 intracellular domain, and a 4-1BB domain as described herein. In several embodiments, this receptorcomplex is encoded by a nucleic acid molecule comprising a sequenceobtained from a combination of sequences disclosed herein, or comprisesan amino acid sequence obtained from a combination of sequencesdisclosed herein. In several embodiments, the encoding nucleic acidsequence, or the amino acid sequence, comprises a sequence in accordancewith one or more SEQ ID NOS as described herein, such as those includedherein as examples of constituent parts. In several embodiments, theencoding nucleic acid sequence, or the amino acid sequence, comprises asequence that shares at least about 90%, at least about 94%, at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,or at least about 99%, sequence identity, homology and/or functionalequivalence with a sequence resulting from the combination one or moreSEQ ID NOS as described herein.

In several embodiments, there is provided a polynucleotide encoding ananti-CD19moiety/CD8 alpha hinge/CD3 TM/CD16/4-1BB/2A/mIL-15 chimericantigen receptor complex (see FIG. 2, CD19-39_10b). The polynucleotidecomprises or is composed of an anti-CD19 moiety, a CD8a hinge, a CD8atransmembrane domain, a CD16 intracellular domain, a 4-1 BB domain, a 2Acleavage site, and an mIL-15 domain as described herein. In severalembodiments, this receptor complex is encoded by a nucleic acid moleculecomprising a sequence obtained from a combination of sequences disclosedherein, or comprises an amino acid sequence obtained from a combinationof sequences disclosed herein. In several embodiments, the encodingnucleic acid sequence, or the amino acid sequence, comprises a sequencein accordance with one or more SEQ ID NOS as described herein, such asthose included herein as examples of constituent parts. In severalembodiments, the encoding nucleic acid sequence, or the amino acidsequence, comprises a sequence that shares at least about 90%, at leastabout 94%, at least about 95%, at least about 96%, at least about 97%,at least about 98%, or at least about 99%, sequence identity, homologyand/or functional equivalence with a sequence resulting from thecombination one or more SEQ ID NOS as described herein.

In several embodiments, there is provided a polynucleotide encoding ananti-CD19moiety/NKG2D Extracellular Domain/CD8hinge-CD8TM/OX40/CD3zetachimeric antigen receptor complex (see FIG. 2, CD19/NKG2D-1a). Thepolynucleotide comprises or is composed of an anti-CD19 moiety, an NKG2Dextracellular domain (either full length or a fragment), a CD8a hinge, aCD8a transmembrane domain, an OX40 domain, and a CD3zeta domain asdescribed herein. In several embodiments, this receptor complex isencoded by a nucleic acid molecule comprising a sequence obtained from acombination of sequences disclosed herein, or comprises an amino acidsequence obtained from a combination of sequences disclosed herein. Inseveral embodiments, the encoding nucleic acid sequence, or the aminoacid sequence, comprises a sequence in accordance with one or more SEQID NOS as described herein, such as those included herein as examples ofconstituent parts. In several embodiments, the encoding nucleic acidsequence, or the amino acid sequence, comprises a sequence that sharesat least about 90%, at least about 94%, at least about 95%, at leastabout 96%, at least about 97%, at least about 98%, or at least about99%, sequence identity, homology and/or functional equivalence with asequence resulting from the combination one or more SEQ ID NOS asdescribed herein.

In several embodiments, there is provided a polynucleotide encoding ananti-CD19moiety/NKG2D EC Domain/CD8hinge-CD8TM/OX40/CD3zeta/2A/mIL-15chimeric antigen receptor complex (see FIG. 2, CD19/NKG2D-1b). Thepolynucleotide comprises or is composed of an anti-CD19 moiety, an NKG2Dextracellular domain (either full length or a fragment), a CD8a hinge, aCD8a transmembrane domain, an OX40 domain, a CD3zeta domain, a 2Acleavage site, and an mIL-15 domain as described herein. In severalembodiments, this receptor complex is encoded by a nucleic acid moleculecomprising a sequence obtained from a combination of sequences disclosedherein, or comprises an amino acid sequence obtained from a combinationof sequences disclosed herein. In several embodiments, the encodingnucleic acid sequence, or the amino acid sequence, comprises a sequencein accordance with one or more SEQ ID NOS as described herein, such asthose included herein as examples of constituent parts. In severalembodiments, the encoding nucleic acid sequence, or the amino acidsequence, comprises a sequence that shares at least about 90%, at leastabout 94%, at least about 95%, at least about 96%, at least about 97%,at least about 98%, or at least about 99%, sequence identity, homologyand/or functional equivalence with a sequence resulting from thecombination one or more SEQ ID NOS as described herein.

In several embodiments, there is provided a polynucleotide encoding ananti-CD19moiety/CD8hinge/CD8TM/4-1BB/CD3zeta/mbIL15 chimeric antigenreceptor complex (see FIG. 3A, NK19). The polynucleotide comprises or iscomposed of an anti-CD19 scFv, a CD8a hinge, a CD8a transmembranedomain, a 4-1BB domain, and a CD3zeta domain. In several embodiments,this receptor complex is encoded by a nucleic acid molecule having thesequence of SEQ ID NO: 85. In several embodiments, a nucleic acidsequence encoding an NK19 chimeric antigen receptor comprises a sequencethat shares at least about 90%, at least about 94%, at least about 95%,at least about 96%, at least about 97%, at least about 98%, or at leastabout 99%, sequence identity, homology and/or functional equivalencewith SEQ ID NO: 85. In several embodiments, the chimeric receptorcomprises the amino acid sequence of SEQ ID NO: 86. In severalembodiments, a NK19 chimeric antigen receptor comprises an amino acidsequence that shares at least about 90%, at least about 94%, at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,or at least about 99%, sequence identity, homology and/or functionalequivalence with SEQ ID NO: 86. Schematically depicted and used inseveral embodiments, there is provided an NK19 construct that lacks anmbIL15 domain (FIG. 3A, NK19 opt.)

In several embodiments, there is provided a polynucleotide encoding ananti-CD19moiety/CD8hinge/CD8TM/OX40/CD3zeta chimeric antigen receptorcomplex (see FIG. 3A, NK19-1a). The polynucleotide comprises or iscomposed of an anti-CD19 scFv, a CD8a hinge, a CD8a transmembranedomain, an OX40 domain, and a CD3zeta domain. In several embodiments,the chimeric antigen receptor further comprises mbIL15 (see FIG. 3A,NK19-1b). In such embodiments, the polynucleotide comprises or iscomposed of an anti-CD19 scFv, a CD8a hinge, a CD8a transmembranedomain, an OX40 domain, a CD3zeta domain, a 2A cleavage site, and anmbIL-15 domain as described herein. In several embodiments, thisreceptor complex is encoded by a nucleic acid molecule having thesequence of SEQ ID NO: 59. In several embodiments, a nucleic acidsequence encoding an NK19 chimeric antigen receptor comprises a sequencethat shares at least about 90%, at least about 94%, at least about 95%,at least about 96%, at least about 97%, at least about 98%, or at leastabout 99%, sequence identity, homology and/or functional equivalencewith SEQ ID NO: 59. In several embodiments, the chimeric receptorcomprises the amino acid sequence of SEQ ID NO: 60. In severalembodiments, a NK19 chimeric antigen receptor comprises an amino acidsequence that shares at least about 90%, at least about 94%, at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,or at least about 99%, sequence identity, homology and/or functionalequivalence with SEQ ID NO: 60. In several embodiments, the CD19 scFvdoes not comprise a Flag tag.

In several embodiments, there is provided a polynucleotide encoding ananti-CD19moiety/CD8hinge/CD28TM/CD28/CD3zeta chimeric antigen receptorcomplex (see FIG. 3A, NK19-2a). The polynucleotide comprises or iscomposed of an anti-CD19 scFv, a CD8a hinge, a CD28 transmembranedomain, CD28 signaling domain, and a CD3zeta domain. In severalembodiments, the chimeric antigen receptor further comprises mbIL15 (seeFIG. 3A, NK19-2b). In such embodiments, the polynucleotide comprises oris composed of an anti-CD19 scFv, a CD8a hinge, a CD28 transmembranedomain, CD28 signaling domain, a CD3zeta domain a 2A cleavage site, andan mbIL-15 domain as described herein. In several embodiments, thisreceptor complex is encoded by a nucleic acid molecule having thesequence of SEQ ID NO: 61. In several embodiments, a nucleic acidsequence encoding an NK19 chimeric antigen receptor comprises a sequencethat shares at least about 90%, at least about 94%, at least about 95%,at least about 96%, at least about 97%, at least about 98%, or at leastabout 99%, sequence identity, homology and/or functional equivalencewith SEQ ID NO: 61. In several embodiments, the chimeric receptorcomprises the amino acid sequence of SEQ ID NO: 62. In severalembodiments, a NK19 chimeric antigen receptor comprises an amino acidsequence that shares at least about 90%, at least about 94%, at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,or at least about 99%, sequence identity, homology and/or functionalequivalence with SEQ ID NO: 62. In several embodiments, the CD19 scFvdoes not comprise a Flag tag.

In several embodiments, there is provided a polynucleotide encoding ananti-CD19moiety/CD8hinge/CD8aTM/ICOS/CD3zeta chimeric antigen receptorcomplex (see FIG. 3A, NK19-3a). The polynucleotide comprises or iscomposed of an anti-CD19 scFv, a CD8a hinge, a CD8a transmembranedomain, inducible costimulator (ICOS) signaling domain, and a CD3zetadomain. In several embodiments, the chimeric antigen receptor furthercomprises mbIL15 (see FIG. 3A, NK19-3b). In such embodiments, thepolynucleotide comprises or is composed of an anti-CD19 scFv, a CD8ahinge, a CD8a transmembrane domain, inducible costimulator (ICOS)signaling domain, a CD3zeta domain, a 2A cleavage site, and an mbIL-15domain as described herein. In several embodiments, this receptorcomplex is encoded by a nucleic acid molecule having the sequence of SEQID NO: 63. In several embodiments, a nucleic acid sequence encoding anNK19 chimeric antigen receptor comprises a sequence that shares at leastabout 90%, at least about 94%, at least about 95%, at least about 96%,at least about 97%, at least about 98%, or at least about 99%, sequenceidentity, homology and/or functional equivalence with SEQ ID NO: 63. Inseveral embodiments, the chimeric receptor comprises the amino acidsequence of SEQ ID NO: 64. In several embodiments, a NK19 chimericantigen receptor comprises an amino acid sequence that shares at leastabout 90%, at least about 94%, at least about 95%, at least about 96%,at least about 97%, at least about 98%, or at least about 99%, sequenceidentity, homology and/or functional equivalence with SEQ ID NO: 64. Inseveral embodiments, the CD19 scFv does not comprise a Flag tag.

In several embodiments, there is provided a polynucleotide encoding ananti-CD19moiety/CD8hinge/CD8aTM/CD28/4-1BB/CD3zeta chimeric antigenreceptor complex (see FIG. 3A, NK19-4a). The polynucleotide comprises oris composed of an anti-CD19 scFv, a CD8a hinge, a CD8a transmembranedomain, a CD28 signaling domain, a 4-1 BB signaling domain, and aCD3zeta domain. In several embodiments, the chimeric antigen receptorfurther comprises mbIL15 (see FIG. 3A, NK19-4b). In such embodiments,the polynucleotide comprises or is composed of an anti-CD19 scFv, a CD8ahinge, a CD8a transmembrane domain, a CD28 signaling domain, a 4-1 BBsignaling domain, a CD3zeta domain, a 2A cleavage site, and an mbIL-15domain as described herein. In several embodiments, this receptorcomplex is encoded by a nucleic acid molecule having the sequence of SEQID NO: 65. In several embodiments, a nucleic acid sequence encoding anNK19 chimeric antigen receptor comprises a sequence that shares at leastabout 90%, at least about 94%, at least about 95%, at least about 96%,at least about 97%, at least about 98%, or at least about 99%, sequenceidentity, homology and/or functional equivalence with SEQ ID NO: 65. Inseveral embodiments, the chimeric receptor comprises the amino acidsequence of SEQ ID NO: 66. In several embodiments, a NK19 chimericantigen receptor comprises an amino acid sequence that shares at leastabout 90%, at least about 94%, at least about 95%, at least about 96%,at least about 97%, at least about 98%, or at least about 99%, sequenceidentity, homology and/or functional equivalence with SEQ ID NO: 66. Inseveral embodiments, the CD19 scFv does not comprise a Flag tag.

In several embodiments, there is provided a polynucleotide encoding ananti-CD19moiety/CD8hinge/NKG2DTM/OX40/CD3zeta chimeric antigen receptorcomplex (see FIG. 3B, NK19-5a). The polynucleotide comprises or iscomposed of an anti-CD19 scFv, a CD8a hinge, a NKG2D transmembranedomain, an OX40 signaling domain, and a CD3zeta domain. In severalembodiments, the chimeric antigen receptor further comprises mbIL15 (seeFIG. 3B, NK19-5b). In such embodiments, the polynucleotide comprises oris composed of an anti-CD19 scFv, a CD8a hinge, a NKG2D transmembranedomain, an OX40 signaling domain, a CD3zeta domain, a 2A cleavage site,and an mbIL-15 domain as described herein. In several embodiments, thisreceptor complex is encoded by a nucleic acid molecule having thesequence of SEQ ID NO: 67. In several embodiments, a nucleic acidsequence encoding an NK19 chimeric antigen receptor comprises a sequencethat shares at least about 90%, at least about 94%, at least about 95%,at least about 96%, at least about 97%, at least about 98%, or at leastabout 99%, sequence identity, homology and/or functional equivalencewith SEQ ID NO: 67. In several embodiments, the chimeric receptorcomprises the amino acid sequence of SEQ ID NO: 68. In severalembodiments, a NK19 chimeric antigen receptor comprises an amino acidsequence that shares at least about 90%, at least about 94%, at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,or at least about 99%, sequence identity, homology and/or functionalequivalence with SEQ ID NO: 68. In several embodiments, the CD19 scFvdoes not comprise a Flag tag.

In several embodiments, there is provided a polynucleotide encoding ananti-CD19moiety/CD8hinge/CD8aTM/CD40/CD3zeta chimeric antigen receptorcomplex (see FIG. 3B, NK19-6a). The polynucleotide comprises or iscomposed of an anti-CD19 scFv variable heavy chain, a CD8a hinge, a CD8atransmembrane domain, a CD40 signaling domain, and a CD3zeta domain. Inseveral embodiments, the chimeric antigen receptor further comprisesmbIL15 (see FIG. 3B, NK19-6b). In such embodiments, the polynucleotidecomprises or is composed of an anti-CD19 scFv variable heavy chain, aCD8a hinge, a CD8a transmembrane domain, a CD40 signaling domain, aCD3zeta domain, a 2A cleavage site, and an mbIL-15 domain as describedherein. In several embodiments, this receptor complex is encoded by anucleic acid molecule having the sequence of SEQ ID NO: 69. In severalembodiments, a nucleic acid sequence encoding an NK19 chimeric antigenreceptor comprises a sequence that shares at least about 90%, at leastabout 94%, at least about 95%, at least about 96%, at least about 97%,at least about 98%, or at least about 99%, sequence identity, homologyand/or functional equivalence with SEQ ID NO: 69. In severalembodiments, the chimeric receptor comprises the amino acid sequence ofSEQ ID NO: 70. In several embodiments, a NK19 chimeric antigen receptorcomprises an amino acid sequence that shares at least about 90%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99%, sequence identity,homology and/or functional equivalence with SEQ ID NO: 70. In severalembodiments, the CD19 scFv does not comprise a Flag tag.

In several embodiments, there is provided a polynucleotide encoding ananti-CD19moiety/CD8hinge/CD8aTM/OX40/CD3zeta/2A/EGFRt chimeric antigenreceptor complex (see FIG. 3B, NK19-7a). The polynucleotide comprises oris composed of an anti-CD19 scFv, a CD8a hinge, a CD8a transmembranedomain, an OX40 signaling domain, a CD3zeta domain, a 2A cleavage side,and a truncated version of the epidermal growth factor receptor (EGFRt).In several embodiments, the chimeric antigen receptor further comprisesmbIL15 (see FIG. 3B, NK19-7b). In such embodiments, the polynucleotidecomprises or is composed of an anti-CD19 scFv, a CD8a hinge, a CD8atransmembrane domain, an OX40 signaling domain, a CD3zeta domain, a 2Acleavage side, a truncated version of the epidermal growth factorreceptor (EGFRt), an additional 2A cleavage site, and an mbIL-15 domainas described herein. In several embodiments, this receptor complex isencoded by a nucleic acid molecule having the sequence of SEQ ID NO: 71.In several embodiments, a nucleic acid sequence encoding an NK19chimeric antigen receptor comprises a sequence that shares at leastabout 90%, at least about 94%, at least about 95%, at least about 96%,at least about 97%, at least about 98%, or at least about 99%, sequenceidentity, homology and/or functional equivalence with SEQ ID NO: 71. Inseveral embodiments, the chimeric receptor comprises the amino acidsequence of SEQ ID NO: 72. In several embodiments, a NK19 chimericantigen receptor comprises an amino acid sequence that shares at leastabout 90%, at least about 94%, at least about 95%, at least about 96%,at least about 97%, at least about 98%, or at least about 99%, sequenceidentity, homology and/or functional equivalence with SEQ ID NO: 72. Inseveral embodiments, the CD19 scFv does not comprise a Flag tag.

In several embodiments, there is provided a polynucleotide encoding ananti-CD19moiety/CD8hinge/CD8aTM/CD40/CD3zeta chimeric antigen receptorcomplex (see FIG. 3B, NK19-8a). The polynucleotide comprises or iscomposed of an anti-CD19 scFv variable light chain, a CD8a hinge, a CD8atransmembrane domain, a CD40 signaling domain, and a CD3zeta domain. Inseveral embodiments, the chimeric antigen receptor further comprisesmbIL15 (see FIG. 3B, NK19-7b). In such embodiments, the polynucleotidecomprises or is composed of an anti-CD19 scFv variable light chain, aCD8a hinge, a CD8a transmembrane domain, a CD40 signaling domain, aCD3zeta domain, a 2A cleavage site, and an mbIL-15 domain as describedherein. In several embodiments, this receptor complex is encoded by anucleic acid molecule having the sequence of SEQ ID NO: 73. In severalembodiments, a nucleic acid sequence encoding an NK19 chimeric antigenreceptor comprises a sequence that shares at least about 90%, at leastabout 94%, at least about 95%, at least about 96%, at least about 97%,at least about 98%, or at least about 99%, sequence identity, homologyand/or functional equivalence with SEQ ID NO: 73. In severalembodiments, the chimeric receptor comprises the amino acid sequence ofSEQ ID NO: 74. In several embodiments, a NK19 chimeric antigen receptorcomprises an amino acid sequence that shares at least about 90%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99%, sequence identity,homology and/or functional equivalence with SEQ ID NO: 74. In severalembodiments, the CD19 scFv does not comprise a Flag tag.

In several embodiments, there is provided a polynucleotide encoding ananti-CD19moiety/CD8hinge/CD8aTM/CD40/CD3zeta chimeric antigen receptorcomplex (see FIG. 3B, NK19-8a). The polynucleotide comprises or iscomposed of an anti-CD19 scFv variable light chain, a CD8a hinge, a CD8atransmembrane domain, a CD40 signaling domain, and a CD3zeta domain. Inseveral embodiments, the chimeric antigen receptor further comprisesmbIL15 (see FIG. 3B, NK19-7b). In such embodiments, the polynucleotidecomprises or is composed of an anti-CD19 scFv variable light chain, aCD8a hinge, a CD8a transmembrane domain, a CD40 signaling domain, aCD3zeta domain, a 2A cleavage site, and an mbIL-15 domain as describedherein. In several embodiments, this receptor complex is encoded by anucleic acid molecule having the sequence of SEQ ID NO: 73. In severalembodiments, a nucleic acid sequence encoding an NK19 chimeric antigenreceptor comprises a sequence that shares at least about 90%, at leastabout 94%, at least about 95%, at least about 96%, at least about 97%,at least about 98%, or at least about 99%, sequence identity, homologyand/or functional equivalence with SEQ ID NO: 73. In severalembodiments, the chimeric receptor comprises the amino acid sequence ofSEQ ID NO: 74. In several embodiments, a NK19 chimeric antigen receptorcomprises an amino acid sequence that shares at least about 90%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99%, sequence identity,homology and/or functional equivalence with SEQ ID NO: 74. In severalembodiments, the CD19 scFv does not comprise a Flag tag.

In several embodiments, there is provided a polynucleotide encoding ananti-CD19moiety/CD8hinge/CD8aTM/CD27/CD3zeta chimeric antigen receptorcomplex (see FIG. 3C, NK19-9a). The polynucleotide comprises or iscomposed of an anti-CD19 scFv, a CD8a hinge, a CD8a transmembranedomain, a CD27 signaling domain, and a CD3zeta domain. In severalembodiments, the chimeric antigen receptor further comprises mbIL15 (seeFIG. 3C, NK19-9b). In such embodiments, the polynucleotide comprises oris composed of an anti-CD19 scFv, a CD8a hinge, a CD8a transmembranedomain, a CD27 signaling domain, a CD3zeta domain, a 2A cleavage site,and an mbIL-15 domain as described herein. In several embodiments, thisreceptor complex is encoded by a nucleic acid molecule having thesequence of SEQ ID NO: 75. In several embodiments, a nucleic acidsequence encoding an NK19 chimeric antigen receptor comprises a sequencethat shares at least about 90%, at least about 94%, at least about 95%,at least about 96%, at least about 97%, at least about 98%, or at leastabout 99%, sequence identity, homology and/or functional equivalencewith SEQ ID NO: 75. In several embodiments, the chimeric receptorcomprises the amino acid sequence of SEQ ID NO: 76. In severalembodiments, a NK19 chimeric antigen receptor comprises an amino acidsequence that shares at least about 90%, at least about 94%, at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,or at least about 99%, sequence identity, homology and/or functionalequivalence with SEQ ID NO: 76. In several embodiments, the CD19 scFvdoes not comprise a Flag tag.

In several embodiments, there is provided a polynucleotide encoding ananti-CD19moiety/CD8hinge/CD8aTM/CD70/CD3zeta chimeric antigen receptorcomplex (see FIG. 3C, NK19-10a). The polynucleotide comprises or iscomposed of an anti-CD19 scFv, a CD8a hinge, a CD8a transmembranedomain, a CD70 signaling domain, and a CD3zeta domain. In severalembodiments, the chimeric antigen receptor further comprises mbIL15 (seeFIG. 3C, NK19-10b). In such embodiments, the polynucleotide comprises oris composed of an anti-CD19 scFv, a CD8a hinge, a CD8a transmembranedomain, a CD70 signaling domain, a CD3zeta domain, a 2A cleavage site,and an mbIL-15 domain as described herein. In several embodiments, thisreceptor complex is encoded by a nucleic acid molecule having thesequence of SEQ ID NO: 77. In several embodiments, a nucleic acidsequence encoding an NK19 chimeric antigen receptor comprises a sequencethat shares at least about 90%, at least about 94%, at least about 95%,at least about 96%, at least about 97%, at least about 98%, or at leastabout 99%, sequence identity, homology and/or functional equivalencewith SEQ ID NO: 77. In several embodiments, the chimeric receptorcomprises the amino acid sequence of SEQ ID NO: 78. In severalembodiments, a NK19 chimeric antigen receptor comprises an amino acidsequence that shares at least about 90%, at least about 94%, at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,or at least about 99%, sequence identity, homology and/or functionalequivalence with SEQ ID NO: 78. In several embodiments, the CD19 scFvdoes not comprise a Flag tag.

In several embodiments, there is provided a polynucleotide encoding ananti-CD19moiety/CD8hinge/CD8aTM/CD161/CD3zeta chimeric antigen receptorcomplex (see FIG. 3C, NK19-11a). The polynucleotide comprises or iscomposed of an anti-CD19 scFv, a CD8a hinge, a CD8a transmembranedomain, a CD161 signaling domain, and a CD3zeta domain. In severalembodiments, the chimeric antigen receptor further comprises mbIL15 (seeFIG. 3C, NK19-11b). In such embodiments, the polynucleotide comprises oris composed of an anti-CD19 scFv, a CD8a hinge, a CD8a transmembranedomain, a CD161 signaling domain, a CD3zeta domain, a 2A cleavage site,and an mbIL-15 domain as described herein. In several embodiments, thisreceptor complex is encoded by a nucleic acid molecule having thesequence of SEQ ID NO: 79. In several embodiments, a nucleic acidsequence encoding an NK19 chimeric antigen receptor comprises a sequencethat shares at least about 90%, at least about 94%, at least about 95%,at least about 96%, at least about 97%, at least about 98%, or at leastabout 99%, sequence identity, homology and/or functional equivalencewith SEQ ID NO: 79. In several embodiments, the chimeric receptorcomprises the amino acid sequence of SEQ ID NO: 80. In severalembodiments, a NK19 chimeric antigen receptor comprises an amino acidsequence that shares at least about 90%, at least about 94%, at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,or at least about 99%, sequence identity, homology and/or functionalequivalence with SEQ ID NO: 80. In several embodiments, the CD19 scFvdoes not comprise a Flag tag.

In several embodiments, there is provided a polynucleotide encoding ananti-CD19moiety/CD8hinge/CD8aTM/CD40L/CD3zeta chimeric antigen receptorcomplex (see FIG. 3C, NK19-12a). The polynucleotide comprises or iscomposed of an anti-CD19 scFv, a CD8a hinge, a CD8a transmembranedomain, a CD40L signaling domain, and a CD3zeta domain. In severalembodiments, the chimeric antigen receptor further comprises mbIL15 (seeFIG. 3C, NK19-12b). In such embodiments, the polynucleotide comprises oris composed of an anti-CD19 scFv, a CD8a hinge, a CD8a transmembranedomain, a CD40L signaling domain, a CD3zeta domain, a 2A cleavage site,and an mbIL-15 domain as described herein. In several embodiments, thisreceptor complex is encoded by a nucleic acid molecule having thesequence of SEQ ID NO: 81. In several embodiments, a nucleic acidsequence encoding an NK19 chimeric antigen receptor comprises a sequencethat shares at least about 90%, at least about 94%, at least about 95%,at least about 96%, at least about 97%, at least about 98%, or at leastabout 99%, sequence identity, homology and/or functional equivalencewith SEQ ID NO: 81. In several embodiments, the chimeric receptorcomprises the amino acid sequence of SEQ ID NO: 82. In severalembodiments, a NK19 chimeric antigen receptor comprises an amino acidsequence that shares at least about 90%, at least about 94%, at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,or at least about 99%, sequence identity, homology and/or functionalequivalence with SEQ ID NO: 82. In several embodiments, the CD19 scFvdoes not comprise a Flag tag.

In several embodiments, there is provided a polynucleotide encoding ananti-CD19moiety/CD8hinge/CD8aTM/CD44/CD3zeta chimeric antigen receptorcomplex (see FIG. 3C, NK19-13). The polynucleotide comprises or iscomposed of an anti-CD19 scFv, a CD8a hinge, a CD8a transmembranedomain, a CD44 signaling domain, and a CD3zeta domain. In severalembodiments, the chimeric antigen receptor further comprises mbIL15 (seeFIG. 3C, NK19-13b). In such embodiments, the polynucleotide comprises oris composed of an anti-CD19 scFv, a CD8a hinge, a CD8a transmembranedomain, a CD44 signaling domain, a CD3zeta domain, a 2A cleavage site,and an mbIL-15 domain as described herein. In several embodiments, thisreceptor complex is encoded by a nucleic acid molecule having thesequence of SEQ ID NO: 83. In several embodiments, a nucleic acidsequence encoding an NK19 chimeric antigen receptor comprises a sequencethat shares at least about 90%, at least about 94%, at least about 95%,at least about 96%, at least about 97%, at least about 98%, or at leastabout 99%, sequence identity, homology and/or functional equivalencewith SEQ ID NO: 83. In several embodiments, the chimeric receptorcomprises the amino acid sequence of SEQ ID NO: 84. In severalembodiments, a NK19 chimeric antigen receptor comprises an amino acidsequence that shares at least about 90%, at least about 94%, at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,or at least about 99%, sequence identity, homology and/or functionalequivalence with SEQ ID NO: 84. In several embodiments, the CD19 scFvdoes not comprise a Flag tag.

In several embodiments, there is provided a polynucleotide encoding aFlag-tag humanized anti-CD19moiety/CD8hinge/CD8TM/OX40/CD3zeta chimericantigen receptor complex (see FIG. 3D, NK19H-1a). The polynucleotidecomprises or is composed of an anti-CD19 scFv that has been humanizedand comprises a first humanized light chain and a first humanized heavychain (L1/H1), and comprises a Flag tag, a CD8a hinge, a CD8atransmembrane domain, an OX40 signaling domain, and a CD3zeta domain. Inseveral embodiments, the chimeric antigen receptor further comprisesmbIL15 (see FIG. 3D, NK19H-1b). In such embodiments, the polynucleotidecomprises or is composed of an anti-CD19 scFv that has been humanizedand comprises a first humanized light chain and a first humanized heavychain (L1/H1), and comprises a Flag tag, a CD8a hinge, a CD8atransmembrane domain, an OX40 signaling domain, and a CD3zeta domain, a2A cleavage site, and an mbIL-15 domain as described herein. In severalembodiments, this receptor complex is encoded by a nucleic acid moleculehaving the sequence of SEQ ID NO: 160. In several embodiments, a nucleicacid sequence encoding an NK19 chimeric antigen receptor comprises asequence that shares at least about 90%, at least about 94%, at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,or at least about 99%, sequence identity, homology and/or functionalequivalence with SEQ ID NO: 160. In several embodiments, the chimericreceptor comprises the amino acid sequence of SEQ ID NO: 161. In severalembodiments, a NK19 chimeric antigen receptor comprises an amino acidsequence that shares at least about 90%, at least about 94%, at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,or at least about 99%, sequence identity, homology and/or functionalequivalence with SEQ ID NO: 161.

In several embodiments, there is provided a polynucleotide encoding aFlag-tag humanized anti-CD19moiety/CD8hinge/CD8TM/OX40/CD3zeta chimericantigen receptor complex (see FIG. 3D, NK19H-2a). The polynucleotidecomprises or is composed of an anti-CD19 scFv that has been humanized,and comprises a second humanized light chain and a first humanized heavychain (L2/H1), and comprises a Flag tag, a CD8a hinge, a CD8atransmembrane domain, an OX40 signaling domain, and a CD3zeta domain. Inseveral embodiments, the chimeric antigen receptor further comprisesmbIL15 (see FIG. 3D, NK19H-2b). In such embodiments, the polynucleotidecomprises or is composed of an anti-CD19 scFv that has been humanizedcomprises a second humanized light chain and a first humanized heavychain (L2/H1), and comprises a Flag tag, a CD8a hinge, a CD8atransmembrane domain, an OX40 signaling domain, a CD3zeta domain, a 2Acleavage site, and an mbIL-15 domain as described herein. In severalembodiments, this receptor complex is encoded by a nucleic acid moleculehaving the sequence of SEQ ID NO: 162. In several embodiments, a nucleicacid sequence encoding an NK19 chimeric antigen receptor comprises asequence that shares at least about 90%, at least about 94%, at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,or at least about 99%, sequence identity, homology and/or functionalequivalence with SEQ ID NO: 162. In several embodiments, the chimericreceptor comprises the amino acid sequence of SEQ ID NO: 163. In severalembodiments, a NK19 chimeric antigen receptor comprises an amino acidsequence that shares at least about 90%, at least about 94%, at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,or at least about 99%, sequence identity, homology and/or functionalequivalence with SEQ ID NO: 162.

In several embodiments, there is provided a polynucleotide encoding aFlag-tag humanized anti-CD19moiety/CD8hinge/CD8TM/OX40/CD3zeta chimericantigen receptor complex (see FIG. 3D, NK19H-3a). The polynucleotidecomprises or is composed of an anti-CD19 scFv that has been humanizedand comprises a third humanized light chain and a first humanized heavychain (L3/H1), and comprises a Flag tag, a CD8a hinge, a CD8atransmembrane domain, an OX40 signaling domain, and a CD3zeta domain. Inseveral embodiments, the chimeric antigen receptor further comprisesmbIL15 (see FIG. 3D, NK19H-3b). In such embodiments, the polynucleotidecomprises or is composed of an anti-CD19 scFv that has been humanizedand comprises a third humanized light chain and a first humanized heavychain (L3/H1), and comprises a Flag tag, a CD8a hinge, a CD8atransmembrane domain, an OX40 signaling domain, and a CD3zeta domain, a2A cleavage site, and an mbIL-15 domain as described herein. In severalembodiments, this receptor complex is encoded by a nucleic acid moleculehaving the sequence of SEQ ID NO: 164. In several embodiments, a nucleicacid sequence encoding an NK19 chimeric antigen receptor comprises asequence that shares at least about 90%, at least about 94%, at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,or at least about 99%, sequence identity, homology and/or functionalequivalence with SEQ ID NO: 164. In several embodiments, the chimericreceptor comprises the amino acid sequence of SEQ ID NO: 165. In severalembodiments, a NK19 chimeric antigen receptor comprises an amino acidsequence that shares at least about 90%, at least about 94%, at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,or at least about 99%, sequence identity, homology and/or functionalequivalence with SEQ ID NO: 165.

In several embodiments, there is provided a polynucleotide encoding aFlag-tag humanized anti-CD19moiety/CD8hinge/CD8TM/OX40/CD3zeta chimericantigen receptor complex (see FIG. 3D, NK19H-4a). The polynucleotidecomprises or is composed of an anti-CD19 scFv that has been humanizedand comprises a first humanized light chain and a second humanized heavychain (L1/H2), and comprises a Flag tag, a CD8a hinge, a CD8atransmembrane domain, an OX40 signaling domain, and a CD3zeta domain. Inseveral embodiments, the chimeric antigen receptor further comprisesmbIL15 (see FIG. 3D, NK19H-4b). In such embodiments, the polynucleotidecomprises or is composed of an anti-CD19 scFv that has been humanizedand comprises a first humanized light chain and a second humanized heavychain (L1/H2), and comprises a Flag tag, a CD8a hinge, a CD8atransmembrane domain, an OX40 signaling domain, and a CD3zeta domain, a2A cleavage site, and an mbIL-15 domain as described herein. In severalembodiments, this receptor complex is encoded by a nucleic acid moleculehaving the sequence of SEQ ID NO: 166. In several embodiments, a nucleicacid sequence encoding an NK19 chimeric antigen receptor comprises asequence that shares at least about 90%, at least about 94%, at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,or at least about 99%, sequence identity, homology and/or functionalequivalence with SEQ ID NO: 166. In several embodiments, the chimericreceptor comprises the amino acid sequence of SEQ ID NO: 167. In severalembodiments, a NK19 chimeric antigen receptor comprises an amino acidsequence that shares at least about 90%, at least about 94%, at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,or at least about 99%, sequence identity, homology and/or functionalequivalence with SEQ ID NO: 167.

In several embodiments, there is provided a polynucleotide encoding aFlag-tag humanized anti-CD19moiety/CD8hinge/CD8TM/OX40/CD3zeta chimericantigen receptor complex (see FIG. 3E, NK19H-5a). The polynucleotidecomprises or is composed of an anti-CD19 scFv that has been humanizedand comprises a second humanized light chain and a second humanizedheavy chain (L2/H2), and comprises a Flag tag, a CD8a hinge, a CD8atransmembrane domain, an OX40 signaling domain, and a CD3zeta domain. Inseveral embodiments, the chimeric antigen receptor further comprisesmbIL15 (see FIG. 3E, NK19H-5b). In such embodiments, the polynucleotidecomprises or is composed of an anti-CD19 scFv that has been humanizedand comprises a second humanized light chain and a second humanizedheavy chain (L2/H2), and comprises a Flag tag, a CD8a hinge, a CD8atransmembrane domain, an OX40 signaling domain, and a CD3zeta domain, a2A cleavage site, and an mbIL-15 domain as described herein. In severalembodiments, this receptor complex is encoded by a nucleic acid moleculehaving the sequence of SEQ ID NO: 168. In several embodiments, a nucleicacid sequence encoding an NK19 chimeric antigen receptor comprises asequence that shares at least about 90%, at least about 94%, at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,or at least about 99%, sequence identity, homology and/or functionalequivalence with SEQ ID NO: 168. In several embodiments, the chimericreceptor comprises the amino acid sequence of SEQ ID NO: 169. In severalembodiments, a NK19 chimeric antigen receptor comprises an amino acidsequence that shares at least about 90%, at least about 94%, at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,or at least about 99%, sequence identity, homology and/or functionalequivalence with SEQ ID NO: 169.

In several embodiments, there is provided a polynucleotide encoding aFlag-tag humanized anti-CD19moiety/CD8hinge/CD8TM/OX40/CD3zeta chimericantigen receptor complex (see FIG. 3E, NK19H-6a). The polynucleotidecomprises or is composed of an anti-CD19 scFv that has been humanizedand comprises a third humanized light chain and a second humanized heavychain (L3/H2), and comprises a Flag tag, a CD8a hinge, a CD8atransmembrane domain, an OX40 signaling domain, and a CD3zeta domain. Inseveral embodiments, the chimeric antigen receptor further comprisesmbIL15 (see FIG. 3E, NK19H-6b). In such embodiments, the polynucleotidecomprises or is composed of an anti-CD19 scFv that has been humanizedand comprises a third humanized light chain and a second humanized heavychain (L3/H2) and comprises a Flag tag, a CD8a hinge, a CD8atransmembrane domain, an OX40 signaling domain, and a CD3zeta domain, a2A cleavage site, and an mbIL-15 domain as described herein. In severalembodiments, this receptor complex is encoded by a nucleic acid moleculehaving the sequence of SEQ ID NO: 170. In several embodiments, a nucleicacid sequence encoding an NK19 chimeric antigen receptor comprises asequence that shares at least about 90%, at least about 94%, at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,or at least about 99%, sequence identity, homology and/or functionalequivalence with SEQ ID NO: 170. In several embodiments, the chimericreceptor comprises the amino acid sequence of SEQ ID NO: 171. In severalembodiments, a NK19 chimeric antigen receptor comprises an amino acidsequence that shares at least about 90%, at least about 94%, at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,or at least about 99%, sequence identity, homology and/or functionalequivalence with SEQ ID NO: 171.

In several embodiments, there is provided a polynucleotide encoding anFlag-tag humanized anti-CD19moiety/CD8hinge/CD8TM/OX40/CD3zeta chimericantigen receptor complex (see FIG. 3E, NK19H-7a). The polynucleotidecomprises or is composed of an anti-CD19 scFv that has been humanizedand comprises a first humanized light chain and a third humanized heavychain (L1/H3), and comprises a Flag tag, a CD8a hinge, a CD8atransmembrane domain, an OX40 signaling domain, and a CD3zeta domain. Inseveral embodiments, the chimeric antigen receptor further comprisesmbIL15 (see FIG. 3E, NK19H-7b). In such embodiments, the polynucleotidecomprises or is composed of an anti-CD19 scFv that has been humanizedand comprises a first humanized light chain and a third humanized heavychain (L1/H3), and comprises a Flag tag, a CD8a hinge, a CD8atransmembrane domain, an OX40 signaling domain, and a CD3zeta domain, a2A cleavage side, a truncated version of the epidermal growth factorreceptor (EGFRt), an additional 2A cleavage site, and an mbIL-15 domainas described herein. In several embodiments, this receptor complex isencoded by a nucleic acid molecule having the sequence of SEQ ID NO:172. In several embodiments, a nucleic acid sequence encoding an NK19chimeric antigen receptor comprises a sequence that shares at leastabout 90%, at least about 94%, at least about 95%, at least about 96%,at least about 97%, at least about 98%, or at least about 99%, sequenceidentity, homology and/or functional equivalence with SEQ ID NO: 172. Inseveral embodiments, the chimeric receptor comprises the amino acidsequence of SEQ ID NO: 173. In several embodiments, a NK19 chimericantigen receptor comprises an amino acid sequence that shares at leastabout 90%, at least about 94%, at least about 95%, at least about 96%,at least about 97%, at least about 98%, or at least about 99%, sequenceidentity, homology and/or functional equivalence with SEQ ID NO: 174.

In several embodiments, there is provided a polynucleotide encoding aFlag-tag humanized anti-CD19moiety/CD8hinge/CD8TM/OX40/CD3zeta chimericantigen receptor complex (see FIG. 3E, NK19H-8a). The polynucleotidecomprises or is composed of an anti-CD19 scFv that has been humanizedand comprises a second humanized light chain and a third humanized heavychain (L2/H3), and comprises a Flag tag, a CD8a hinge, a CD8atransmembrane domain, an OX40 signaling domain, and a CD3zeta domain. Inseveral embodiments, the chimeric antigen receptor further comprisesmbIL15 (see FIG. 3E, NKH19-8b). In such embodiments, the polynucleotidecomprises or is composed of an anti-CD19 scFv that has been humanizedand comprises a second humanized light chain and a third humanized heavychain (L2/H3), and comprises a Flag tag, a CD8a hinge, a CD8atransmembrane domain, an OX40 signaling domain, and a CD3zeta domain, a2A cleavage site, and an mbIL-15 domain as described herein. In severalembodiments, this receptor complex is encoded by a nucleic acid moleculehaving the sequence of SEQ ID NO: 174. In several embodiments, a nucleicacid sequence encoding an NK19 chimeric antigen receptor comprises asequence that shares at least about 90%, at least about 94%, at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,or at least about 99%, sequence identity, homology and/or functionalequivalence with SEQ ID NO: 174. In several embodiments, the chimericreceptor comprises the amino acid sequence of SEQ ID NO: 175. In severalembodiments, a NK19 chimeric antigen receptor comprises an amino acidsequence that shares at least about 90%, at least about 94%, at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,or at least about 99%, sequence identity, homology and/or functionalequivalence with SEQ ID NO: 175.

In several embodiments, there is provided a polynucleotide encoding aFlag-tag humanized anti-CD19moiety/CD8hinge/CD8TM/OX40/CD3zeta chimericantigen receptor complex (see FIG. 3E, NK19H-9a). The polynucleotidecomprises or is composed of an anti-CD19 scFv that has been humanizedand comprises a third humanized light chain and a third humanized heavychain (L3/H3), and comprises a Flag tag, a CD8a hinge, a CD8atransmembrane domain, an OX40 signaling domain, and a CD3zeta domain. Inseveral embodiments, the chimeric antigen receptor further comprisesmbIL15 (see FIG. 3E, NKH19-9b). In such embodiments, the polynucleotidecomprises or is composed of an anti-CD19 scFv that has been humanizedand comprises a third humanized light chain and a third humanized heavychain (L3/H3), and comprises a Flag tag, a CD8a hinge, a CD8atransmembrane domain, an OX40 signaling domain, and a CD3zeta domain, a2A cleavage site, and an mbIL-15 domain as described herein. In severalembodiments, this receptor complex is encoded by a nucleic acid moleculehaving the sequence of SEQ ID NO: 176. In several embodiments, a nucleicacid sequence encoding an NK19 chimeric antigen receptor comprises asequence that shares at least about 90%, at least about 94%, at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,or at least about 99%, sequence identity, homology and/or functionalequivalence with SEQ ID NO: 176. In several embodiments, the chimericreceptor comprises the amino acid sequence of SEQ ID NO: 177. In severalembodiments, a NK19 chimeric antigen receptor comprises an amino acidsequence that shares at least about 90%, at least about 94%, at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,or at least about 99%, sequence identity, homology and/or functionalequivalence with SEQ ID NO: 177.

In several embodiments, there is provided a polynucleotide encoding aFlag-tag humanized anti-CD19moiety/CD8hinge/CD8TM/OX40/CD3zeta chimericantigen receptor complex (see FIG. 3E, NKH19-10a). The polynucleotidecomprises or is composed of an anti-CD19 scFv that has been humanizedand comprises a first humanized light chain and a fourth humanized heavychain (L1/H4), and comprises a Flag tag, a CD8a hinge, a CD8atransmembrane domain, an OX40 signaling domain, and a CD3zeta domain. Inseveral embodiments, the chimeric antigen receptor further comprisesmbIL15 (see FIG. 3E, NK19H-10b). In such embodiments, the polynucleotidecomprises or is composed of an anti-CD19 scFv that has been humanizedand comprises a first humanized light chain and a fourth humanized heavychain (L1/H4), and comprises a Flag tag, a CD8a hinge, a CD8atransmembrane domain, an OX40 signaling domain, and a CD3zeta domain, a2A cleavage site, and an mbIL-15 domain as described herein. In severalembodiments, this receptor complex is encoded by a nucleic acid moleculehaving the sequence of SEQ ID NO: 178. In several embodiments, a nucleicacid sequence encoding an NK19 chimeric antigen receptor comprises asequence that shares at least about 90%, at least about 94%, at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,or at least about 99%, sequence identity, homology and/or functionalequivalence with SEQ ID NO: 178. In several embodiments, the chimericreceptor comprises the amino acid sequence of SEQ ID NO: 179. In severalembodiments, a NK19 chimeric antigen receptor comprises an amino acidsequence that shares at least about 90%, at least about 94%, at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,or at least about 99%, sequence identity, homology and/or functionalequivalence with SEQ ID NO: 179.

In several embodiments, there is provided a polynucleotide encoding aFlag-tag humanized anti-CD19moiety/CD8hinge/CD8TM/OX40/CD3zeta chimericantigen receptor complex (see FIG. 3F, NK19H-11a). The polynucleotidecomprises or is composed of an anti-CD19 scFv that has been humanizedand comprises a second humanized light chain and a fourth humanizedheavy chain (L2/H4), and comprises a Flag tag, a CD8a hinge, a CD8atransmembrane domain, an OX40 signaling domain, and a CD3zeta domain. Inseveral embodiments, the chimeric antigen receptor further comprisesmbIL15 (see FIG. 3F, NK19H-11b). In such embodiments, the polynucleotidecomprises or is composed of an anti-CD19 scFv that has been humanizedand comprises a second humanized light chain and a fourth humanizedheavy chain (L2/H4), and comprises a Flag tag, a CD8a hinge, a CD8atransmembrane domain, an OX40 signaling domain, and a CD3zeta domain, a2A cleavage site, and an mbIL-15 domain as described herein. In severalembodiments, this receptor complex is encoded by a nucleic acid moleculehaving the sequence of SEQ ID NO: 180. In several embodiments, a nucleicacid sequence encoding an NK19 chimeric antigen receptor comprises asequence that shares at least about 90%, at least about 94%, at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,or at least about 99%, sequence identity, homology and/or functionalequivalence with SEQ ID NO: 180. In several embodiments, the chimericreceptor comprises the amino acid sequence of SEQ ID NO: 181. In severalembodiments, a NK19 chimeric antigen receptor comprises an amino acidsequence that shares at least about 90%, at least about 94%, at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,or at least about 99%, sequence identity, homology and/or functionalequivalence with SEQ ID NO: 181.

In several embodiments, there is provided a polynucleotide encoding aFlag-tag, humanized anti-CD19moiety/CD8hinge/CD8TM/OX40/CD3zeta chimericantigen receptor complex (see FIG. 3F NK19H-12a). The polynucleotidecomprises or is composed of an anti-CD19 scFv that has been humanizedand comprises a third humanized light chain and a fourth humanized heavychain (L3/H4), and comprises a Flag tag, a CD8a hinge, a CD8atransmembrane domain, an OX40 signaling domain, and a CD3zeta domain. Inseveral embodiments, the chimeric antigen receptor further comprisesmbIL15 (see FIG. 3F, NK19H-12b). In such embodiments, the polynucleotidecomprises or is composed of an anti-CD19 scFv that has been humanizedand comprises a third humanized light chain and a fourth humanized heavychain (L3/H4), and comprises a Flag tag, a CD8a hinge, a CD8atransmembrane domain, an OX40 signaling domain, and a CD3zeta domain, a2A cleavage site, and an mbIL-15 domain as described herein. In severalembodiments, this receptor complex is encoded by a nucleic acid moleculehaving the sequence of SEQ ID NO: 182. In several embodiments, a nucleicacid sequence encoding an NK19 chimeric antigen receptor comprises asequence that shares at least about 90%, at least about 94%, at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,or at least about 99%, sequence identity, homology and/or functionalequivalence with SEQ ID NO: 182. In several embodiments, the chimericreceptor comprises the amino acid sequence of SEQ ID NO: 183. In severalembodiments, a NK19 chimeric antigen receptor comprises an amino acidsequence that shares at least about 90%, at least about 94%, at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,or at least about 99%, sequence identity, homology and/or functionalequivalence with SEQ ID NO: 183.

In several embodiments, there is provided a polynucleotide encodingchimeric antigen receptor that comprises a Flag-tag, humanizedanti-CD19moiety and multiple co-stimulatory domains. For example, aschematic architecture is anti-CD19 moiety/transmembranedomain/co-stimulatory domain 1/co-stimulatory domain 2/co-stimulatorydomain 3/signaling domain. The co-stimulatory domains vary in order,depending on the embodiment. For example, in several embodiments theco-stimulatory domains (“CSD”) may be positioned as: CSD1/CSD2,CSD2/CSD1, CSD1/CSD2/CSD3, CSD1/CSD2/CSD3, CSD3/CSD2/CSD1, etc. Inseveral embodiments, there is provided a polynucleotide encoding aFlag-tag, humanizedanti-CD19moiety/CD8hinge/CD8aTM/CD44/OX40/CD27/CD3zeta chimeric antigenreceptor complex (see FIG. 3F, NK19H-13a). The polynucleotide comprisesor is composed of an anti-CD19 scFv that has been humanized andcomprises a Flag tag, a CD8a hinge, a CD8a transmembrane domain, a CD44co-stimulatory domain, an OX40 co-stimulatory domain, a CD27co-stimulatory domain, and a CD3zeta domain. In several embodiments, thechimeric antigen receptor further comprises mbIL15 (see FIG. 3F,NK19H-13b). In such embodiments, the polynucleotide comprises or iscomposed of an anti-CD19 scFv that has been humanized and comprises aFlag tag, a CD8a hinge, a CD8a transmembrane domain, a CD44co-stimulatory domain, an OX40 co-stimulatory domain, a CD27co-stimulatory domain, a CD3zeta domain, a 2A cleavage site, and anmbIL-15 domain as described herein.

In several embodiments, there is provided a polynucleotide encoding ahumanized anti-CD19moiety/CD8hinge/CD8TM/OX40/CD3zeta chimeric antigenreceptor complex (see FIG. 3G, NK19H-NF-1a). The polynucleotidecomprises or is composed of an anti-CD19 scFv that has been humanizedand comprises a first humanized light chain and a first humanized heavychain (L1/H1), a CD8a hinge, a CD8a transmembrane domain, an OX40signaling domain, and a CD3zeta domain. In several embodiments, thechimeric antigen receptor further comprises mbIL15 (see FIG. 3G,NK19H-NF-1b). In such embodiments, the polynucleotide comprises or iscomposed of an anti-CD19 scFv that has been humanized and comprises afirst humanized light chain and a first humanized heavy chain (L1/H1), aCD8a hinge, a CD8a transmembrane domain, an OX40 signaling domain, and aCD3zeta domain, a 2A cleavage site, and an mbIL-15 domain as describedherein. In several embodiments, this receptor complex is encoded by anucleic acid molecule having the sequence of SEQ ID NO: 184. In severalembodiments, a nucleic acid sequence encoding an NK19 chimeric antigenreceptor comprises a sequence that shares at least about 90%, at leastabout 94%, at least about 95%, at least about 96%, at least about 97%,at least about 98%, or at least about 99%, sequence identity, homologyand/or functional equivalence with SEQ ID NO: 184. In severalembodiments, the chimeric receptor comprises the amino acid sequence ofSEQ ID NO: 185. In several embodiments, a NK19 chimeric antigen receptorcomprises an amino acid sequence that shares at least about 90%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99%, sequence identity,homology and/or functional equivalence with SEQ ID NO: 185.

In several embodiments, there is provided a polynucleotide encoding ahumanized anti-anti-CD19moiety/CD8hinge/CD8TM/OX40/CD3zeta chimericantigen receptor complex (see FIG. 3G, NK19H-NF-2a). The polynucleotidecomprises or is composed of an anti-CD19 scFv that has been humanizedand comprises a second humanized light chain and a first humanized heavychain (L2/H1), a CD8a hinge, a CD8a transmembrane domain, an OX40signaling domain, and a CD3zeta domain. In several embodiments, thechimeric antigen receptor further comprises mbIL15 (see FIG. 3G,NK19H-NF-2b). In such embodiments, the polynucleotide comprises or iscomposed of an anti-CD19 scFv that has been humanized and comprises asecond humanized light chain and a first humanized heavy chain (L2/H1),a CD8a hinge, a CD8a transmembrane domain, an OX40 signaling domain, anda CD3zeta domain, a 2A cleavage site, and an mbIL-15 domain as describedherein. In several embodiments, this receptor complex is encoded by anucleic acid molecule having the sequence of SEQ ID NO: 186. In severalembodiments, a nucleic acid sequence encoding an NK19 chimeric antigenreceptor comprises a sequence that shares at least about 90%, at leastabout 94%, at least about 95%, at least about 96%, at least about 97%,at least about 98%, or at least about 99%, sequence identity, homologyand/or functional equivalence with SEQ ID NO: 186. In severalembodiments, the chimeric receptor comprises the amino acid sequence ofSEQ ID NO: 187. In several embodiments, a NK19 chimeric antigen receptorcomprises an amino acid sequence that shares at least about 90%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99%, sequence identity,homology and/or functional equivalence with SEQ ID NO: 187.

In several embodiments, there is provided a polynucleotide encoding ahumanized anti-anti-CD19moiety/CD8hinge/CD8TM/OX40/CD3zeta chimericantigen receptor complex (see FIG. 3G, NK19H-NF-3a). The polynucleotidecomprises or is composed of an anti-CD19 scFv that has been humanizedand comprises a third humanized light chain and a first humanized heavychain (L3/H1), a CD8a hinge, a CD8a transmembrane domain, an OX40signaling domain, and a CD3zeta domain. In several embodiments, thechimeric antigen receptor further comprises mbIL15 (see FIG. 3G,NK19H-NF-3b). In such embodiments, the polynucleotide comprises or iscomposed of an anti-CD19 scFv that has been humanized and comprises athird humanized light chain and a first humanized heavy chain (L3/H1), aCD8a hinge, a CD8a transmembrane domain, an OX40 signaling domain, and aCD3zeta domain, a 2A cleavage site, and an mbIL-15 domain as describedherein. In several embodiments, this receptor complex is encoded by anucleic acid molecule having the sequence of SEQ ID NO: 188. In severalembodiments, a nucleic acid sequence encoding an NK19 chimeric antigenreceptor comprises a sequence that shares at least about 90%, at leastabout 94%, at least about 95%, at least about 96%, at least about 97%,at least about 98%, or at least about 99%, sequence identity, homologyand/or functional equivalence with SEQ ID NO: 188. In severalembodiments, the chimeric receptor comprises the amino acid sequence ofSEQ ID NO: 189. In several embodiments, a NK19 chimeric antigen receptorcomprises an amino acid sequence that shares at least about 90%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99%, sequence identity,homology and/or functional equivalence with SEQ ID NO: 189.

In several embodiments, there is provided a polynucleotide encoding ahumanized anti-anti-CD19moiety/CD8hinge/CD8TM/OX40/CD3zeta chimericantigen receptor complex (see FIG. 3G, NK19H-NF-4a). The polynucleotidecomprises or is composed of an anti-CD19 scFv that has been humanizedand comprises a first humanized light chain and a second humanized heavychain (L1/H2), a CD8a hinge, a CD8a transmembrane domain, an OX40signaling domain, and a CD3zeta domain. In several embodiments, thechimeric antigen receptor further comprises mbIL15 (see FIG. 3G,NK19H-NF-4b). In such embodiments, the polynucleotide comprises or iscomposed of an anti-CD19 scFv that has been humanized and comprises afirst humanized light chain and a second humanized heavy chain (L1/H2),a CD8a hinge, a CD8a transmembrane domain, an OX40 signaling domain, anda CD3zeta domain, a 2A cleavage site, and an mbIL-15 domain as describedherein. In several embodiments, this receptor complex is encoded by anucleic acid molecule having the sequence of SEQ ID NO: 190. In severalembodiments, a nucleic acid sequence encoding an NK19 chimeric antigenreceptor comprises a sequence that shares at least about 90%, at leastabout 94%, at least about 95%, at least about 96%, at least about 97%,at least about 98%, or at least about 99%, sequence identity, homologyand/or functional equivalence with SEQ ID NO: 190. In severalembodiments, the chimeric receptor comprises the amino acid sequence ofSEQ ID NO: 191. In several embodiments, a NK19 chimeric antigen receptorcomprises an amino acid sequence that shares at least about 90%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99%, sequence identity,homology and/or functional equivalence with SEQ ID NO: 191.

In several embodiments, there is provided a polynucleotide encoding ahumanized anti-CD19moiety/CD8hinge/CD8TM/OX40/CD3zeta chimeric antigenreceptor complex (see FIG. 3H, NK19H-NF-5a). The polynucleotidecomprises or is composed of an anti-CD19 scFv that has been humanizedand comprises a second humanized light chain and a second humanizedheavy chain (L2/H2), a CD8a hinge, a CD8a transmembrane domain, an OX40signaling domain, and a CD3zeta domain. In several embodiments, thechimeric antigen receptor further comprises mbIL15 (see FIG. 3H,NK19H-NF-5b). In such embodiments, the polynucleotide comprises or iscomposed of an anti-CD19 scFv that has been humanized and comprises asecond humanized light chain and a second humanized heavy chain (L2/H2),a CD8a hinge, a CD8a transmembrane domain, an OX40 signaling domain, anda CD3zeta domain, a 2A cleavage site, and an mbIL-15 domain as describedherein. In several embodiments, this receptor complex is encoded by anucleic acid molecule having the sequence of SEQ ID NO: 192. In severalembodiments, a nucleic acid sequence encoding an NK19 chimeric antigenreceptor comprises a sequence that shares at least about 90%, at leastabout 94%, at least about 95%, at least about 96%, at least about 97%,at least about 98%, or at least about 99%, sequence identity, homologyand/or functional equivalence with SEQ ID NO: 192. In severalembodiments, the chimeric receptor comprises the amino acid sequence ofSEQ ID NO: 193. In several embodiments, a NK19 chimeric antigen receptorcomprises an amino acid sequence that shares at least about 90%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99%, sequence identity,homology and/or functional equivalence with SEQ ID NO: 193.

In several embodiments, there is provided a polynucleotide encoding ahumanized anti-CD19moiety/CD8hinge/CD8TM/OX40/CD3zeta chimeric antigenreceptor complex (see FIG. 3H, NK19H-NF-6a). The polynucleotidecomprises or is composed of an anti-CD19 scFv that has been humanizedand comprises a third humanized light chain and a second humanized heavychain (L3/H2), a CD8a hinge, a CD8a transmembrane domain, an OX40signaling domain, and a CD3zeta domain. In several embodiments, thechimeric antigen receptor further comprises mbIL15 (see FIG. 3H,NK19H-NF-6b). In such embodiments, the polynucleotide comprises or iscomposed of an anti-CD19 scFv that has been humanized and comprises athird humanized light chain and a second humanized heavy chain (L3/H2),a CD8a hinge, a CD8a transmembrane domain, an OX40 signaling domain, anda CD3zeta domain, a 2A cleavage site, and an mbIL-15 domain as describedherein. In several embodiments, this receptor complex is encoded by anucleic acid molecule having the sequence of SEQ ID NO: 194. In severalembodiments, a nucleic acid sequence encoding an NK19 chimeric antigenreceptor comprises a sequence that shares at least about 90%, at leastabout 94%, at least about 95%, at least about 96%, at least about 97%,at least about 98%, or at least about 99%, sequence identity, homologyand/or functional equivalence with SEQ ID NO: 194. In severalembodiments, the chimeric receptor comprises the amino acid sequence ofSEQ ID NO: 195. In several embodiments, a NK19 chimeric antigen receptorcomprises an amino acid sequence that shares at least about 90%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99%, sequence identity,homology and/or functional equivalence with SEQ ID NO: 195.

In several embodiments, there is provided a polynucleotide encoding ahumanized anti-CD19moiety/CD8hinge/CD8TM/OX40/CD3zeta chimeric antigenreceptor complex (see FIG. 3G, NK19H-NF-7a). The polynucleotidecomprises or is composed of an anti-CD19 scFv that has been humanizedand comprises a first humanized light chain and a third humanized heavychain (L1/H3), a CD8a hinge, a CD8a transmembrane domain, an OX40signaling domain, and a CD3zeta domain. In several embodiments, thechimeric antigen receptor further comprises mbIL15 (see FIG. 3H,NK19H-NF-7b). In such embodiments, the polynucleotide comprises or iscomposed of an anti-CD19 scFv that has been humanized and comprises afirst humanized light chain and a third humanized heavy chain (L1/H3), aCD8a hinge, a CD8a transmembrane domain, an OX40 signaling domain, aCD3zeta domain, a 2A cleavage site, and an mbIL-15 domain as describedherein. In several embodiments, this receptor complex is encoded by anucleic acid molecule having the sequence of SEQ ID NO: 196. In severalembodiments, a nucleic acid sequence encoding an NK19 chimeric antigenreceptor comprises a sequence that shares at least about 90%, at leastabout 94%, at least about 95%, at least about 96%, at least about 97%,at least about 98%, or at least about 99%, sequence identity, homologyand/or functional equivalence with SEQ ID NO: 196. In severalembodiments, the chimeric receptor comprises the amino acid sequence ofSEQ ID NO: 197. In several embodiments, a NK19 chimeric antigen receptorcomprises an amino acid sequence that shares at least about 90%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99%, sequence identity,homology and/or functional equivalence with SEQ ID NO: 197.

In several embodiments, there is provided a polynucleotide encoding ahumanized anti-CD19moiety/CD8hinge/CD8TM/OX40/CD3zeta chimeric antigenreceptor complex (see FIG. 3H, NK19H-NF-8a). The polynucleotidecomprises or is composed of an anti-CD19 scFv that has been humanizedand comprises a second humanized light chain and a third humanized heavychain (L2/H3), a CD8a hinge, a CD8a transmembrane domain, an OX40signaling domain, and a CD3zeta domain. In several embodiments, thechimeric antigen receptor further comprises mbIL15 (see FIG. 3H,NKH19-NF-8b). In such embodiments, the polynucleotide comprises or iscomposed of an anti-CD19 scFv variable light chain that has beenhumanized and comprises a second humanized light chain and a thirdhumanized heavy chain (L2/H3), a CD8a hinge, a CD8a transmembranedomain, an OX40 signaling domain, and a CD3zeta domain, a 2A cleavagesite, and an mbIL-15 domain as described herein. In several embodiments,this receptor complex is encoded by a nucleic acid molecule having thesequence of SEQ ID NO: 198. In several embodiments, a nucleic acidsequence encoding an NK19 chimeric antigen receptor comprises a sequencethat shares at least about 90%, at least about 94%, at least about 95%,at least about 96%, at least about 97%, at least about 98%, or at leastabout 99%, sequence identity, homology and/or functional equivalencewith SEQ ID NO: 198. In several embodiments, the chimeric receptorcomprises the amino acid sequence of SEQ ID NO: 199. In severalembodiments, a NK19 chimeric antigen receptor comprises an amino acidsequence that shares at least about 90%, at least about 94%, at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,or at least about 99%, sequence identity, homology and/or functionalequivalence with SEQ ID NO: 199.

In several embodiments, there is provided a polynucleotide encoding ahumanized anti-anti-CD19moiety/CD8hinge/CD8TM/OX40/CD3zeta chimericantigen receptor complex (see FIG. 3H, NK19H-NF-9a). The polynucleotidecomprises or is composed of an anti-CD19 scFv that has been humanizedand comprises a third humanized light chain and a third humanized heavychain (L3/H3), a CD8a hinge, a CD8a transmembrane domain, an OX40signaling domain, and a CD3zeta domain. In several embodiments, thechimeric antigen receptor further comprises mbIL15 (see FIG. 3H,NKH19-NF-9b). In such embodiments, the polynucleotide comprises or iscomposed of an anti-CD19 scFv that has been humanized and comprises athird humanized light chain and a third humanized heavy chain (L3/H3), aCD8a hinge, a CD8a transmembrane domain, an OX40 signaling domain, and aCD3zeta domain, a 2A cleavage site, and an mbIL-15 domain as describedherein. In several embodiments, this receptor complex is encoded by anucleic acid molecule having the sequence of SEQ ID NO: 200. In severalembodiments, a nucleic acid sequence encoding an NK19 chimeric antigenreceptor comprises a sequence that shares at least about 90%, at leastabout 94%, at least about 95%, at least about 96%, at least about 97%,at least about 98%, or at least about 99%, sequence identity, homologyand/or functional equivalence with SEQ ID NO: 200. In severalembodiments, the chimeric receptor comprises the amino acid sequence ofSEQ ID NO: 201. In several embodiments, a NK19 chimeric antigen receptorcomprises an amino acid sequence that shares at least about 90%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99%, sequence identity,homology and/or functional equivalence with SEQ ID NO: 201.

In several embodiments, there is provided a polynucleotide encoding ahumanized anti-CD19moiety/CD8hinge/CD8TM/OX40/CD3zeta chimeric antigenreceptor complex (see FIG. 3H, NKH19-NF-10a). The polynucleotidecomprises or is composed of an anti-CD19 scFv that has been humanizedand comprises a first humanized light chain and a fourth humanized heavychain (L1/H4), a CD8a hinge, a CD8a transmembrane domain, an OX40signaling domain, and a CD3zeta domain. In several embodiments, thechimeric antigen receptor further comprises mbIL15 (see FIG. 3H,NK19H-NF-10b). In such embodiments, the polynucleotide comprises or iscomposed of an anti-CD19 scFv that has been humanized and comprises afirst humanized light chain and a fourth humanized heavy chain (L1/H4),a CD8a hinge, a CD8a transmembrane domain, an OX40 signaling domain, anda CD3zeta domain, a 2A cleavage site, and an mbIL-15 domain as describedherein. In several embodiments, this receptor complex is encoded by anucleic acid molecule having the sequence of SEQ ID NO: 202. In severalembodiments, a nucleic acid sequence encoding an NK19 chimeric antigenreceptor comprises a sequence that shares at least about 90%, at leastabout 94%, at least about 95%, at least about 96%, at least about 97%,at least about 98%, or at least about 99%, sequence identity, homologyand/or functional equivalence with SEQ ID NO: 202. In severalembodiments, the chimeric receptor comprises the amino acid sequence ofSEQ ID NO: 203. In several embodiments, a NK19 chimeric antigen receptorcomprises an amino acid sequence that shares at least about 90%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99%, sequence identity,homology and/or functional equivalence with SEQ ID NO: 203.

In several embodiments, there is provided a polynucleotide encoding ahumanized anti-CD19moiety/CD8hinge/CD8TM/OX40/CD3zeta chimeric antigenreceptor complex (see FIG. 3I, NK19H-NF-11a). The polynucleotidecomprises or is composed of an anti-CD19 scFv that has been humanizedand comprises a second humanized light chain and a fourth humanizedheavy chain (L2/H4), a CD8a hinge, a CD8a transmembrane domain, an OX40signaling domain, and a CD3zeta domain. In several embodiments, thechimeric antigen receptor further comprises mbIL15 (see FIG. 3I,NK19H-NF-11b). In such embodiments, the polynucleotide comprises or iscomposed of an anti-CD19 scFv that has been humanized, and comprises asecond humanized light chain and a fourth humanized heavy chain (L2/H4),a CD8a hinge, a CD8a transmembrane domain, an OX40 signaling domain, anda CD3zeta domain, a 2A cleavage site, and an mbIL-15 domain as describedherein. In several embodiments, this receptor complex is encoded by anucleic acid molecule having the sequence of SEQ ID NO: 204. In severalembodiments, a nucleic acid sequence encoding an NK19 chimeric antigenreceptor comprises a sequence that shares at least about 90%, at leastabout 94%, at least about 95%, at least about 96%, at least about 97%,at least about 98%, or at least about 99%, sequence identity, homologyand/or functional equivalence with SEQ ID NO: 204. In severalembodiments, the chimeric receptor comprises the amino acid sequence ofSEQ ID NO: 205. In several embodiments, a NK19 chimeric antigen receptorcomprises an amino acid sequence that shares at least about 90%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99%, sequence identity,homology and/or functional equivalence with SEQ ID NO: 205.

In several embodiments, there is provided a polynucleotide encoding ahumanized anti-CD19moiety/CD8hinge/CD8TM/OX40/CD3zeta chimeric antigenreceptor complex (see FIG. 3I NK19H-12a). The polynucleotide comprisesor is composed of an anti-CD19 scFv that has been humanized andcomprises a third humanized light chain and a fourth humanized heavychain (L3/H4), a CD8a hinge, a CD8a transmembrane domain, an OX40signaling domain, and a CD3zeta domain. In several embodiments, thechimeric antigen receptor further comprises mbIL15 (see FIG. 3I,NK19H-NF-12b). In such embodiments, the polynucleotide comprises or iscomposed of an anti-CD19 scFv that has been humanized and comprises athird humanized light chain and a fourth humanized heavy chain (L3/H4),a CD8a hinge, a CD8a transmembrane domain, an OX40 signaling domain, anda CD3zeta domain, a 2A cleavage site, and an mbIL-15 domain as describedherein. In several embodiments, this receptor complex is encoded by anucleic acid molecule having the sequence of SEQ ID NO: 206. In severalembodiments, a nucleic acid sequence encoding an NK19 chimeric antigenreceptor comprises a sequence that shares at least about 90%, at leastabout 94%, at least about 95%, at least about 96%, at least about 97%,at least about 98%, or at least about 99%, sequence identity, homologyand/or functional equivalence with SEQ ID NO: 206. In severalembodiments, the chimeric receptor comprises the amino acid sequence ofSEQ ID NO: 207. In several embodiments, a NK19 chimeric antigen receptorcomprises an amino acid sequence that shares at least about 90%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99%, sequence identity,homology and/or functional equivalence with SEQ ID NO: 207.

In several embodiments, there is provided a polynucleotide encodingchimeric antigen receptor that comprises a Flag-tag, humanizedanti-CD19moiety and multiple co-stimulatory domains. For example, aschematic architecture is anti-CD19 moiety/transmembranedomain/co-stimulatory domain 1/co-stimulatory domain 2/co-stimulatorydomain 3/signaling domain. The co-stimulatory domains vary in order,depending on the embodiment. For example, in several embodiments theco-stimulatory domains (“CSD”) may be positioned as: CSD1/CSD2,CSD2/CSD1, CSD1/CSD2/CSD3, CSD1/CSD2/CSD3, CSD3/CSD2/CSD1, etc. Inseveral embodiments, there is provided a polynucleotide encoding aFlag-tag, humanizedanti-CD19moiety/CD8hinge/CD8aTM/CD44/OX40/CD27/CD3zeta chimeric antigenreceptor complex (see FIG. 3I, NK19H-NF-13a). The polynucleotidecomprises or is composed of an anti-CD19 scFv that has been humanizedand comprises a Flag tag, a CD8a hinge, a CD8a transmembrane domain, aCD44 co-stimulatory domain, an OX40 co-stimulatory domain, a CD27co-stimulatory domain, and a CD3zeta domain. In several embodiments, thechimeric antigen receptor further comprises mbIL15 (see FIG. 3I,NK19H-NF-13b). In such embodiments, the polynucleotide comprises or iscomposed of an anti-CD19 scFv that has been humanized and comprises aFlag tag, a CD8a hinge, a CD8a transmembrane domain, a CD44co-stimulatory domain, an OX40 co-stimulatory domain, a CD27co-stimulatory domain, a CD3zeta domain, a 2A cleavage site, and anmbIL-15 domain as described herein.

In several embodiments, there is provided a polynucleotide encodingchimeric antigen receptor that comprises a Flag-tag, humanizedanti-CD19moiety and multiple co-stimulatory domains. For example, aschematic architecture is anti-CD19 moiety/transmembranedomain/co-stimulatory domain 1/co-stimulatory domain 2/co-stimulatorydomain 3/signaling domain. The co-stimulatory domains vary in order,depending on the embodiment. For example, in several embodiments theco-stimulatory domains (“CSD”) may be positioned as: CSD1/CSD2,CSD2/CSD1, CSD1/CSD2/CSD3, CSD1/CSD2/CSD3, CSD3/CSD2/CSD1, etc. Inseveral embodiments, there is provided a polynucleotide encoding aFlag-tag, humanizedanti-CD19moiety/CD8hinge/CD8aTM/CD44/OX40/CD27/CD3zeta chimeric antigenreceptor complex (see FIG. 3F, NK19H-13a). The polynucleotide comprisesor is composed of an anti-CD19 scFv that has been humanized andcomprises a Flag tag, a CD8a hinge, a CD8a transmembrane domain, a CD44co-stimulatory domain, an OX40 co-stimulatory domain, a CD27co-stimulatory domain, and a CD3zeta domain. In several embodiments, thechimeric antigen receptor further comprises mbIL15 (see FIG. 3F,NK19H-13b). In such embodiments, the polynucleotide comprises or iscomposed of an anti-CD19 scFv that has been humanized and comprises aFlag tag, a CD8a hinge, a CD8a transmembrane domain, a CD44co-stimulatory domain, an OX40 co-stimulatory domain, a CD27co-stimulatory domain, a CD3zeta domain, a 2A cleavage site, and anmbIL-15 domain as described herein.

It shall be appreciated that, for any receptor construct describedherein, certain sequence variability, extensions, and/or truncations ofthe disclosed sequences may result when combining sequences, as a resultof, for example, ease or efficiency in cloning (e.g., for creation of arestriction site).

Methods of Treatment

Some embodiments relate to a method of treating, ameliorating,inhibiting, or preventing cancer with a cell or immune cell comprising achimeric receptor such as a CD19-directed chimeric receptor. In someembodiments, the method includes treating or preventing cancer. In someembodiments, the method includes administering a therapeuticallyeffective amount of immune cells expressing a CD19-directed chimericreceptor as described herein. Examples of types of cancer that may betreated as such are described herein.

In certain embodiments, treatment of a subject with a geneticallyengineered cell(s) described herein achieves one, two, three, four, ormore of the following effects, including, for example: (i) reduction oramelioration the severity of disease or symptom associated therewith;(ii) reduction in the duration of a symptom associated with a disease;(iii) protection against the progression of a disease or symptomassociated therewith; (iv) regression of a disease or symptom associatedtherewith; (v) protection against the development or onset of a symptomassociated with a disease; (vi) protection against the recurrence of asymptom associated with a disease; (vii) reduction in thehospitalization of a subject; (viii) reduction in the hospitalizationlength; (ix) an increase in the survival of a subject with a disease;(x) a reduction in the number of symptoms associated with a disease;(xi) an enhancement, improvement, supplementation, complementation, oraugmentation of the prophylactic or therapeutic effect(s) of anothertherapy. Administration can be by a variety of routes, including,without limitation, intravenous, intra-arterial, subcutaneous,intramuscular, intrahepatic, intraperitoneal and/or local delivery to anaffected tissue.

Administration and Dosing

Further provided herein are methods of treating a subject having cancer,comprising administering to the subject a composition comprising immunecells (such as NK and/or T cells) engineered to express a cytotoxicreceptor complex as disclosed herein. For example, some embodiments ofthe compositions and methods described herein relate to use of aCD19-directed chimeric receptor, or use of cells expressing theCD19-directed chimeric receptor, for treating a cancer patient. Uses ofsuch engineered immune cells for treating cancer are also provided.

In certain embodiments, treatment of a subject with a geneticallyengineered cell(s) described herein achieves one, two, three, four, ormore of the following effects, including, for example: (i) reduction oramelioration the severity of disease or symptom associated therewith;(ii) reduction in the duration of a symptom associated with a disease;(iii) protection against the progression of a disease or symptomassociated therewith; (iv) regression of a disease or symptom associatedtherewith; (v) protection against the development or onset of a symptomassociated with a disease; (vi) protection against the recurrence of asymptom associated with a disease; (vii) reduction in thehospitalization of a subject; (viii) reduction in the hospitalizationlength; (ix) an increase in the survival of a subject with a disease;(x) a reduction in the number of symptoms associated with a disease;(xi) an enhancement, improvement, supplementation, complementation, oraugmentation of the prophylactic or therapeutic effect(s) of anothertherapy. Each of these comparisons are versus, for example, a differenttherapy for a disease, which includes a cell-based immunotherapy for adisease using cells that do not express the constructs disclosed herein.

Administration can be by a variety of routes, including, withoutlimitation, intravenous, intra-arterial, subcutaneous, intramuscular,intrahepatic, intraperitoneal and/or local delivery to an affectedtissue. Doses of immune cells such as NK and/or T cells can be readilydetermined for a given subject based on their body mass, disease typeand state, and desired aggressiveness of treatment, but range, dependingon the embodiments, from about 10⁵ cells per kg to about 10¹² cells perkg (e.g., 10⁵-10⁷, 10⁷-10¹⁰, 10¹⁰-10¹² and overlapping ranges therein).In one embodiment, a dose escalation regimen is used. In severalembodiments, a range of immune cells such as NK and/or T cells isadministered, for example between about 1×10⁶ cells/kg to about 1×10⁸cells/kg. In several embodiments, the dosage ranges from about 2×10⁵cells/kg to about 2×10⁸ cells/kg, including about 2×10⁶ and 2×10⁷cells/kg. In several embodiments, a dose is determined by the maximumnumber of viable engineered cells at the time of dosing. For example, insome embodiments, a single dose comprises a maximum of between about2×10⁵ and about 2×10⁹ viable engineered cells, including about 2×10⁶,about 2×10⁷, or about 2×10⁸ viable engineered cells. Depending on theembodiment, various types of cancer can be treated. In severalembodiments, hepatocellular carcinoma is treated. Additional embodimentsprovided for herein include treatment or prevention of the followingnon-limiting examples of cancers including, but not limited to, acutelymphoblastic leukemia (ALL), acute myeloid leukemia (AML),adrenocortical carcinoma, Kaposi sarcoma, lymphoma, gastrointestinalcancer, appendix cancer, central nervous system cancer, basal cellcarcinoma, bile duct cancer, bladder cancer, bone cancer, brain tumors(including but not limited to astrocytomas, spinal cord tumors, brainstem glioma, glioblastoma, craniopharyngioma, ependymoblastoma,ependymoma, medulloblastoma, medulloepithelioma), breast cancer,bronchial tumors, Burkitt lymphoma, cervical cancer, colon cancer,chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML),chronic myeloproliferative disorders, ductal carcinoma, endometrialcancer, esophageal cancer, gastric cancer, Hodgkin lymphoma, non-Hodgkinlymphoma, hairy cell leukemia, renal cell cancer, leukemia, oral cancer,nasopharyngeal cancer, liver cancer, lung cancer (including but notlimited to, non-small cell lung cancer, (NSCLC) and small cell lungcancer), pancreatic cancer, bowel cancer, lymphoma, melanoma, ocularcancer, ovarian cancer, pancreatic cancer, prostate cancer, pituitarycancer, uterine cancer, and vaginal cancer.

In some embodiments, also provided herein are nucleic acid and aminoacid sequences that have sequence identity or homology of at least 80%,85%, 90%, 95%, 96%, 97%, 98%, 99% (and ranges therein) as compared withthe respective nucleic acid or amino acid sequences of SEQ ID NOS. 1-207(or combinations of two or more of SEQ ID NOS: 1-207) and that alsoexhibit one or more of the functions as compared with the respective SEQID NOS. 1-207 (or combinations of two or more of SEQ ID NOS: 1-207)including but not limited to, (i) enhanced proliferation, (ii) enhancedactivation, (iii) enhanced cytotoxic activity against cells presentingligands to which NK cells harboring receptors encoded by the nucleicacid and amino acid sequences bind, (iv) enhanced homing to tumor orinfected sites, (v) reduced off target cytotoxic effects, (vi) enhancedsecretion of immunostimulatory cytokines and chemokines (including, butnot limited to IFNg, TNFa, IL-22, CCL3, CCL4, and CCL5), (vii) enhancedability to stimulate further innate and adaptive immune responses, and(viii) combinations thereof.

Additionally, in several embodiments, there are provided amino acidsequences that correspond to any of the nucleic acids disclosed herein,while accounting for degeneracy of the nucleic acid code. Furthermore,those sequences (whether nucleic acid or amino acid) that vary fromthose expressly disclosed herein, but have functional similarity orequivalency are also contemplated within the scope of the presentdisclosure. The foregoing includes mutants, truncations, substitutions,or other types of modifications.

In several embodiments, polynucleotides encoding the disclosed cytotoxicreceptor complexes or CD19-directed chimeric receptors are mRNA. In someembodiments, the polynucleotide is DNA. In some embodiments, thepolynucleotide is operably linked to at least one regulatory element forthe expression of the cytotoxic receptor complex.

Additionally provided, according to several embodiments, is a vectorcomprising the polynucleotide encoding any of the polynucleotidesprovided for herein, wherein the polynucleotides are optionallyoperatively linked to at least one regulatory element for expression ofa cytotoxic receptor complex. In several embodiments, the vector is aretrovirus.

Further provided herein are engineered immune cells (such as NK and/or Tcells) comprising the polynucleotide, vector, or cytotoxic receptorcomplexes as disclosed herein. Further provided herein are compositionscomprising a mixture of engineered immune cells (such as NK cells and/orengineered T cells), each population comprising the polynucleotide,vector, or cytotoxic receptor complexes as disclosed herein.

Doses of immune cells such as NK cells or T cells can be readilydetermined for a given subject based on their body mass, disease typeand state, and desired aggressiveness of treatment, but range, dependingon the embodiments, from about 10⁵ cells per kg to about 10¹² cells perkg (e.g., 10⁵-10⁷, 10⁷-10¹⁰, 10¹⁰-10¹² and overlapping ranges therein).In one embodiment, a dose escalation regimen is used. In severalembodiments, a range of NK cells is administered, for example betweenabout 1×10⁶ cells/kg to about 1×10⁸ cells/kg. Depending on theembodiment, various types of cancer or infection disease can be treated.

Cancer Types

Some embodiments of the compositions and methods described herein relateto administering immune cells comprising a chimeric receptor, such as aCD19-directed chimeric receptor, to a subject with cancer. Variousembodiments provided for herein include treatment or prevention of thefollowing non-limiting examples of cancers. Examples of cancer include,but are not limited to, acute lymphoblastic leukemia (ALL), acutemyeloid leukemia (AML), adrenocortical carcinoma, Kaposi sarcoma,lymphoma, gastrointestinal cancer, appendix cancer, central nervoussystem cancer, basal cell carcinoma, bile duct cancer, bladder cancer,bone cancer, brain tumors (including but not limited to astrocytomas,spinal cord tumors, brain stem glioma, craniopharyngioma,ependymoblastoma, ependymoma, medulloblastoma, medulloepithelioma),breast cancer, bronchial tumors, Burkitt lymphoma, cervical cancer,colon cancer, chronic lymphocytic leukemia (CLL), chronic myelogenousleukemia (CML), chronic myeloproliferative disorders, ductal carcinoma,endometrial cancer, esophageal cancer, gastric cancer, Hodgkin lymphoma,non-Hodgkin lymphoma, hairy cell leukemia, renal cell cancer, leukemia,oral cancer, nasopharyngeal cancer, liver cancer, lung cancer (includingbut not limited to, non-small cell lung cancer, (NSCLC) and small celllung cancer), pancreatic cancer, bowel cancer, lymphoma, melanoma,ocular cancer, ovarian cancer, pancreatic cancer, prostate cancer,pituitary cancer, uterine cancer, and vaginal cancer.

Cancer Targets

Some embodiments of the compositions and methods described herein relateto immune cells comprising a chimeric receptor that targets a cancerantigen. Non-limiting examples of target antigens include: CD5, CD19;CD123; CD22; CD30; CD171; CS1 (also referred to as CD2 subset 1, CRACC,SLAMF7, CD319, and 19A24); C-type lectin-like molecule-1 (CLL-1 orCLECL1); CD33; epidermal growth factor receptor variant III (EGFRviii);ganglioside G2 (GD2); ganglioside GD3(aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(I-4)bDGIcp(I-I)Cer); TNF receptor familymember B cell maturation (BCMA); Tn antigen ((Tn Ag) or(GalNAca-Ser/Thr)); prostate-specific membrane antigen (PSMA); Receptortyrosine kinase-like orphan receptor 1 (ROR1); Fms Like Tyrosine Kinase3 (FLT3); Tumor-associated glycoprotein 72 (TAG72); CD38; CD44v6; aglycosylated CD43 epitope expressed on acute leukemia or lymphoma butnot on hematopoietic progenitors, a glycosylated CD43 epitope expressedon non-hematopoietic cancers, Carcinoembryonic antigen (CEA); Epithelialcell adhesion molecule (EPCAM); B7H3 (CD276); KIT (CD117);Interleukin-13 receptor subunit alpha-2 (IL-13Ra2 or CD213A2);Mesothelin; Interleukin 11 receptor alpha (IL-IIRa); prostate stem cellantigen (PSCA); Protease Serine 21 (Testisin or PRSS21); vascularendothelial growth factor receptor 2 (VEGFR2); Lewis(Y) antigen; CD24;Platelet-derived growth factor receptor beta (PDGFR-beta);Stage-specific embryonic antigen-4 (SSEA-4); CD20; Folate receptor alpha(FRa or FR1); Folate receptor beta (FRb); Receptor tyrosine-proteinkinase ERBB2 (Her2/neu); Mucin 1, cell surface associated (MUC1);epidermal growth factor receptor (EGFR); neural cell adhesion molecule(NCAM); Prostase; prostatic acid phosphatase (PAP); elongation factor 2mutated (ELF2M); Ephrin B2; fibroblast activation protein alpha (FAP);insulin-like growth factor 1 receptor (IGF-I receptor), carbonicanhydrase IX (CAIX); Proteasome (Prosome, Macropain) Subunit, Beta Type,9 (LMP2); glycoprotein 100 (gp100); oncogene fusion protein consistingof breakpoint cluster region (BCR) and Abelson murine leukemia viraloncogene homolog 1 (Abl) (bcr-abl); tyrosinase; ephrin type-A receptor 2(EphA2); sialyl Lewis adhesion molecule (sLe); ganglioside GM3(aNeu5Ac(2-3)bDCIalp(I-4)bDGIcp(I-I)Cer); transglutaminase 5 (TGS5);high molecular weight-melanoma associated antigen (HMWMAA); o-acetyl-GD2ganglioside (OAcGD2); tumor endothelial marker 1 (TEM1/CD248); tumorendothelial marker 7-related (TEM7R); claudin 6 (CLDN6); thyroidstimulating hormone receptor (TSHR); G protein coupled receptor class Cgroup 5, member D (GPRC5D); chromosome X open reading frame 61(CXORF61); CD97; CD179a; anaplastic lymphoma kinase (ALK); Polysialicacid; placenta-specific 1 (PLAC1); hexasaccharide portion of globoHglycoceramide (GloboH); mammary gland differentiation antigen (NY-BR-1);uroplakin 2 (UPK2); Hepatitis A virus cellular receptor 1 (HAVCR1);adrenoceptor beta 3 (ADRB3); pannexin 3 (PANX3); G protein-coupledreceptor 20 (GPR20); lymphocyte antigen 6 complex, locus K 9 (LY6K);Olfactory receptor 51E2 (OR51E2); TCR Gamma Alternate Reading FrameProtein (TARP); Wilms tumor protein (WT1); Cancer/testis antigen 1(NY-ESO-1); Cancer/testis antigen 2 (LAGE-la); Melanoma-associatedantigen 1 (MAGE-A1); ETS translocation-variant gene 6, located onchromosome 12p (ETV6-AML); sperm protein 17 (SPA17); X Antigen Family,Member 1A (XAGE1); angiopoietin-binding cell surface receptor 2 (Tie 2);melanoma cancer testis antigen-1 (MAD-CT-1); melanoma cancer testisantigen-2 (MAD-CT-2); Fos-related antigen 1; tumor protein p53 (p53);p53 mutant; prostein; survivin; telomerase; prostate carcinoma tumorantigen-1 (PCT A-I or Galectin 8), melanoma antigen recognized by Tcells 1 (MelanA or MARTI); Rat sarcoma (Ras) mutant; human Telomerase;reverse transcriptase (hTERT); sarcoma translocation breakpoints;melanoma inhibitor of apoptosis (ML-IAP); ERG (transmembrane protease,serine 2 (TMPRSS2) ETS fusion gene); N-Acetyl glucosaminyl-transferase V(NA17); paired box protein Pax-3 (PAX3); Androgen receptor; Cyclin BI;v-myc avian myelocytomatosis viral oncogene neuroblastoma derivedhomolog (MYCN); Ras Homolog Family Member C (RhoC); Tyrosinase-relatedprotein 2 (TRP-2); Cytochrome P450 IB 1 (CYPIB 1); CCCTC-Binding Factor(Zinc Finger Protein)-Like (BORIS or Brother of the Regulator ofImprinted Sites), Squamous Cell Carcinoma Antigen Recognized By T Cells3 (SART3); Paired box protein Pax-5 (PAXS); proacrosin binding proteinsp32 (OY-TES1); lymphocyte-specific protein tyrosine kinase (LCK); Akinase anchor protein 4 (AKAP-4); synovial sarcoma, X breakpoint 2(SSX2); Receptor for Advanced Gly cation Endproducts (RAGE-1); renalubiquitous 1 (RU1); renal ubiquitous 2 (RU2); legumain; human papillomavirus E6 (HPV E6); human papilloma virus E7 (HPV E7); intestinalcarboxyl esterase; heat shock protein 70-2 mutated (mut hsp70-2); CD79a;CD79b; CD72; Leukocyte-associated immunoglobulin-like receptor 1(LAIR1); Fc fragment of IgA receptor (FCAR or CD89); Leukocyteimmunoglobulin-like receptor subfamily A member 2 (LILRA2); CD300molecule-like family member f (CD300LF); C-type lectin domain family 12member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); EGF-likemodule-containing mucin-like hormone receptor-like 2 (EMR2); lymphocyteantigen 75 (LY75); Glypican-3 (GPC3); Fc receptor-like 5 (FCRLS); andimmunoglobulin lambda-like polypeptide 1 (IGLLI), MPL, Biotin, c-MYCepitope Tag, CD34, LAMP1 TROP2, GFRalpha4, CDH17, CDH6, NYBR1, CDH19,CD200R, Slea (CA19.9; Sialyl Lewis Antigen); Fucosyl-GMI, PTK7, gpNMB,CDH1-CD324, DLL3, CD276/B7H3, ILI IRa, IL13Ra2, CD179b-IGLII,TCRgamma-delta, NKG2D, CD32 (FCGR2A), Tn ag, Timl-/HVCR1, CSF2RA(GM-CSFR-alpha), TGFbetaR2, Lews Ag, TCR-beta1 chain, TCR-beta2 chain,TCR-gamma chain, TCR-delta chain, FITC, Luteinizing hormone receptor(LHR), Follicle stimulating hormone receptor (FSHR), GonadotropinHormone receptor (CGHR or GR), CCR4, GD3, SLAMF6, SLAMF4, HIV1 envelopeglycoprotein, HTLVI-Tax, CMV pp65, EBV-EBNA3c, KSHV K8.1, KSHV-gH,influenza A hemagglutinin (HA), GAD, PDL1, Guanylyl cyclase C (GCC),auto antibody to desmoglein 3 (Dsg3), auto antibody to desmoglein 1(Dsgl), HLA, HLA-A, HLA-A2, HLA-B, HLA-C, HLA-DP, HLA-DM, HLA-DOA,HLA-DOB, HLA-DQ, HLA-DR, HLA-G, IgE, CD99, Ras G12V, Tissue Factor 1(TF1), AFP, GPRC5D, Claudin 18.2 (CLD18A2 or CLDN18A.2)),P-glycoprotein, STEAP1, Livl, Nectin-4, Cripto, gpA33, BST1/CD157, lowconductance chloride channel, and the antigen recognized by TNTantibody.

Additionally, in several embodiments there is provided an immune cellthat expresses a CD19-directed chimeric antigen receptor, the chimericantigen receptor comprising an extracellular anti-CD19 binding moiety,wherein the anti-CD19 binding moiety comprises a heavy chain variable(VH) domain and a and a light chain variable (VL) domain, the VH domaincomprising a VH domain having at least 95% identity to the VH domainamino acid sequence set forth in SEQ ID NO: 33, the VL domain having atleast 95% identity to the VL domain amino acid sequence set forth in SEQID NO: 32, a hinge and/or transmembrane domain, an intracellularsignaling domain, wherein the intracellular signaling domain comprisesan OX40 subdomain, and wherein the cell also expresses membrane-boundinterleukin-15 (mbIL15). In several embodiments, the intracellularsignaling domain further comprises a CD3zeta subdomain. In severalembodiments, the OX40 subdomain comprises the amino acid sequence of SEQID NO: 6 and the CD3zeta subdomain comprises the amino acid sequence ofSEQ ID NO: 7. In several embodiments, the hinge domain comprises a CD8ahinge domain. In several embodiments, the CD8a hinge domain, comprisesthe amino acid sequence of SEQ ID NO: 2. In several embodiments, thembIL15 comprises the amino acid sequence of SEQ ID NO: 12. In severalembodiments, the chimeric receptor further comprises an extracellulardomain of an NKG2D receptor. In several embodiments, the extracellulardomain of the NKG2D receptor comprises a functional fragment of NKG2Dcomprising the amino acid sequence of SEQ ID NO: 26. In severalembodiments, the immune cell is a natural killer (NK) cell. In severalembodiments, the immune cell is a T cell. In several embodiments, thesuch immune cells are administered to a subject in a method of treatingcancer, or are otherwise used to the treatment of cancer, such as in thepreparation of a medicament for the treatment of cancer. In severalembodiments, the cancer is acute lymphocytic leukemia.

In several embodiments there is provided a polynucleotide encoding aCD19-directed chimeric antigen receptor, the chimeric antigen receptorcomprising an extracellular anti-CD19 binding moiety, wherein theanti-CD19 binding moiety comprises a heavy chain variable (VH) domainand a and a light chain variable (VL) domain, the VH domain having atleast 95% identity to the VH domain amino acid sequence set forth in SEQID NO: 33, the VL domain having at least 95% identity to the VL domainamino acid sequence set forth in SEQ ID NO: 32, a hinge and/ortransmembrane domain, an intracellular signaling domain, wherein theintracellular signaling domain comprises an OX40 subdomain, and whereinthe polynucleotide also encodes membrane-bound interleukin-15 (mbIL15).In several embodiments, the intracellular signaling domain furthercomprises a CD3zeta subdomain. In several embodiments, the encoded OX40subdomain comprises the amino acid sequence of SEQ ID NO: 16 and theencoded CD3zeta subdomain comprises the amino acid sequence of SEQ IDNO: 8. In several embodiments, the hinge domain comprises a CD8a hingedomain and comprises the amino acid sequence of SEQ ID NO: 2. In severalembodiments, the encoded mbIL15 comprises the amino acid sequence of SEQID NO: 12. In several embodiments, the chimeric receptor furthercomprises an extracellular domain of an NKG2D receptor. In severalembodiments, the encoded extracellular domain of the NKG2D receptorcomprises a functional fragment of NKG2D comprising the amino acidsequence of SEQ ID NO: 26.

Also provided herein is an immune cell that expresses a CD19-directedchimeric receptor comprising an extracellular anti-CD19 moiety, a hingeand/or transmembrane domain, and an intracellular signaling domain. Inseveral embodiments, the immune cell is an NK cell. In severalembodiments, the immune cell is a T cell. In several embodiments, thehinge domain comprises a CD8a hinge domain or an Ig4 SH domain. Inseveral embodiments, the transmembrane domain comprises a CD8atransmembrane domain, a CD28 transmembrane domain and/or a CD3transmembrane domain. In several embodiments, the signaling domaincomprises an OX40 signaling domain, a 4-1 BB signaling domain, a CD28signaling domain, an NKp80 signaling domain, a CD16 IC signaling domain,a CD3zeta or CD3 ITAM signaling domain, and/or a mIL-15 signalingdomain. In several embodiments, the signaling domain comprises a 2Acleavage domain. In several embodiments, the mIL-15 signaling domain isseparated from the rest or another portion of the CD19-directed chimericreceptor by a 2A cleavage domain. In several embodiments, such immunecells are administered to a subject having cancer in order to treat,inhibit or prevent progression of the cancer.

Provided herein is also an engineered NK or T cell that expresses aCD19-directed chimeric antigen receptor, the chimeric antigen receptorcomprising an extracellular anti-CD19 binding moiety, wherein theanti-CD19 binding moiety comprises a heavy chain variable (VH) domainand a and a light chain variable (VL) domain, the VH domain comprising aVH domain resulting from humanization of the VH domain amino acidsequence set forth in SEQ ID NO: 33, the VL domain comprising a VLdomain resulting from humanization the VL domain amino acid sequence setforth in SEQ ID NO: 32, a hinge and/or transmembrane domain, anintracellular signaling domain, wherein the intracellular signalingdomain comprises an OX40 subdomain, and wherein the cell also expressesmembrane-bound interleukin-15 (mbIL15).

Provided herein is a polynucleotide encoding a CD19-directed chimericantigen receptor, the chimeric antigen receptor comprising anextracellular anti-CD19 binding moiety, wherein the anti-CD19 bindingmoiety comprises a heavy chain variable (VH) domain and a and a lightchain variable (VL) domain, the VH domain comprising a VH domainresulting from humanization of the VH domain amino acid sequence setforth in SEQ ID NO: 33, the VL domain comprising a VL domain resultingfrom humanization the VL domain amino acid sequence set forth in SEQ IDNO: 32; a hinge and/or transmembrane domain,

an intracellular signaling domain, wherein the intracellular signalingdomain comprises an OX40 subdomain, and wherein the polynucleotide alsoencodes membrane-bound interleukin-15 (mbIL15).

Provided here is a polynucleotide encoding a CD19-directed chimericantigen receptor, the chimeric antigen receptor comprising anextracellular anti-CD19 binding moiety, wherein the anti-CD19 bindingmoiety comprises a scFv; a hinge, wherein the hinge is a CD8 alphahinge; a transmembrane domain; and an intracellular signaling domain,wherein the intracellular signaling domain comprises a CD3 zeta ITAM. Inseveral embodiments, the transmembrane domain comprises a CD8 alphatransmembrane domain, an NKG2D transmembrane domain, and/or a CD28transmembrane domain. In several embodiments, the intracellularsignaling domain comprises a CD28 signaling domain, a 4-1 BB signalingdomain, and/or an OX40 domain. In several embodiments, the intracellularsignaling domain may also comprise a domain selected from ICOS, CD70,CD161, CD40L, CD44, and combinations thereof.

Provided herein is a polynucleotide encoding a CD19-directed chimericantigen receptor, the chimeric antigen receptor comprising anextracellular anti-CD19 binding moiety, wherein the anti-CD19 bindingmoiety comprises a variable heavy chain of a scFv or a variable lightchain of a scFv; a hinge, wherein the hinge is a CD8 alpha hinge; atransmembrane domain, wherein the transmembrane domain comprises a CD8alpha transmembrane domain; and an intracellular signaling domain,wherein the intracellular signaling domain comprises a CD3 zeta ITAM. Inseveral embodiments, the polynucleotide also encodes a truncatedepidermal growth factor receptor (EGFRt). In several embodiments, thepolynucleotide also encodes membrane-bound interleukin-15 (mbIL15).Provided herein is an engineered NK or T cell that expresses such aCD19-directed chimeric antigen, as well as methods of treating cancer byadministering such an NK cell or T cell. Also provided for is the use ofsuch polynucleotides in the treatment of cancer, for example, in themanufacture of a medicament for the treatment of cancer.

Provided herein is a polynucleotide encoding a humanized CD19-directedchimeric antigen receptor, the chimeric antigen receptor comprising anextracellular anti-CD19 binding moiety, wherein the anti-CD19 bindingmoiety comprises a heavy chain variable (VH) domain and a and a lightchain variable (VL) domain, the VH domain comprising a VH domainselected from SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, and SEQ IDNO: 123, the VL domain comprising a VL domain selected from SEQ ID NO:117, SEQ ID NO: 118, and SEQ ID NO: 119; a hinge and/or transmembranedomain, an intracellular signaling domain, and wherein thepolynucleotide also encodes membrane-bound interleukin-15 (mbIL15). Inseveral embodiments, the intracellular signaling domain comprises anOX40 subdomain, a CD28 subdomain, an iCOS subdomain, a CD28-41 BBsubdomain, a CD27 subdomain, a CD44 subdomain, or combinations thereof.In several embodiments, the chimeric antigen receptor comprises a hingeand a transmembrane domain, wherein the hinge is a CD8 alpha hinge,wherein the transmembrane domain is either a CD8 alpha or an NKG2Dtransmembrane domain. In several embodiments, the intracellularsignaling domain comprises a CD3zeta domain.

Provided for herein is a polynucleotide encoding a humanized chimericantigen receptor (CAR), wherein the CAR comprises a single chainantibody or single chain antibody fragment which comprises a humanizedanti-CD19 binding domain, a transmembrane domain, a primaryintracellular signaling domain comprising a native intracellularsignaling domain of CD3-zeta, or a functional fragment thereof, and acostimulatory domain comprising a native intracellular signaling domainof a protein selected from the group consisting of OX40, CD27, CD28,ICOS, and 4-1BB, or a functional fragment thereof, wherein saidanti-CD19 binding domain comprises a light chain complementarydetermining region 1 (LC CDR1) of SEQ ID NO: 124, 127, or 130, a lightchain complementary determining region 2 (LC CDR2) of SEQ ID NO: 125,128, or 131, and a light chain complementary determining region 3 (LCCDR3) of SEQ ID NO: 126, 129, or 132, and a heavy chain complementarydetermining region 1 (HC CDR1) of SEQ ID NO: 133, 136, 139, or 142, aheavy chain complementary determining region 2 (HC CDR2) of SEQ ID NO:134, 137, 140, or 143, and a heavy chain complementary determiningregion 3 (HC CDR3) of SEQ ID NO: 135, 138, 141, or 144. In severalembodiments, the polynucleotide further comprises a region encodingmembrane-bound interleukin 15 (mbIL15).

Provided for herein is a polynucleotide encoding a humanizedCD19-directed chimeric antigen receptor, the chimeric antigen receptorcomprising an extracellular anti-CD19 binding moiety, wherein theanti-CD19 binding moiety comprises a humanized scFv sequence comprisinga variable light (VL) domain of SEQ ID NO: 117, a hinge and/ortransmembrane domain, an intracellular signaling domain, and wherein thepolynucleotide also encodes membrane-bound interleukin-15 (mbIL15). Inseveral embodiments, the polynucleotide encodes the humanized chimericantigen receptor of SEQ ID NO: 161, SEQ ID NO: 167, SEQ ID NO: 173, SEQID NO: 179, SEQ ID NO: 185, SEQ ID NO: 191, SEQ ID NO: 197, or SEQ IDNO: 203.

Provided for is a polynucleotide encoding a humanized CD19-directedchimeric antigen receptor, the chimeric antigen receptor comprising anextracellular anti-CD19 binding moiety, wherein the anti-CD19 bindingmoiety comprises a humanized scFv sequence comprising a variable light(VL) domain of SEQ ID NO: 118, a hinge and/or transmembrane domain, anintracellular signaling domain, and wherein the polynucleotide alsoencodes membrane-bound interleukin-15 (mbIL15). In several embodiments,the polynucleotide encodes the humanized chimeric antigen receptor ofSEQ ID NO: 163, SEQ ID NO: 169, SEQ ID NO: 175, SEQ ID NO: 181, SEQ IDNO: 187, SEQ ID NO: 193, SEQ ID NO: 199, or SEQ ID NO: 205.

Provided for is a polynucleotide encoding a humanized CD19-directedchimeric antigen receptor, the chimeric antigen receptor comprising anextracellular anti-CD19 binding moiety, wherein the anti-CD19 bindingmoiety comprises a humanized scFv sequence comprising a variable light(VL) domain of SEQ ID NO: 119, a hinge and/or transmembrane domain, anintracellular signaling domain, and wherein the polynucleotide alsoencodes membrane-bound interleukin-15 (mbIL15). In several embodiments,the polynucleotide encodes the humanized chimeric antigen receptor ofSEQ ID NO: 165, SEQ ID NO: 171, SEQ ID NO: 177, SEQ ID NO: 183, SEQ IDNO: 189, SEQ ID NO: 195, SEQ ID NO: 201, or SEQ ID NO: 207.

Provided for is a polynucleotide encoding a humanized CD19-directedchimeric antigen receptor, the chimeric antigen receptor comprising anextracellular anti-CD19 binding moiety, wherein the anti-CD19 bindingmoiety comprises a humanized scFv sequence comprising a variable heavy(VH) domain of SEQ ID NO: 120, a hinge and/or transmembrane domain, anintracellular signaling domain, and wherein the polynucleotide alsoencodes membrane-bound interleukin-15 (mbIL15). In several embodiments,the polynucleotide encodes the humanized chimeric antigen receptor ofSEQ ID NO: 161, SEQ ID NO: 163, SEQ ID NO: 165, SEQ ID NO: 185, SEQ IDNO: 187, or SEQ ID NO: 189.

Provided for is a polynucleotide encoding a humanized CD19-directedchimeric antigen receptor, the chimeric antigen receptor comprising anextracellular anti-CD19 binding moiety, wherein the anti-CD19 bindingmoiety comprises a humanized scFv sequence comprising a variable heavy(VH) domain of SEQ ID NO: 121, a hinge and/or transmembrane domain, anintracellular signaling domain, and wherein the polynucleotide alsoencodes membrane-bound interleukin-15 (mbIL15). In several embodiments,the polynucleotide encodes the humanized chimeric antigen receptor ofSEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 191, SEQ IDNO: 193, or SEQ ID NO: 195.

Provided for is a polynucleotide encoding a humanized CD19-directedchimeric antigen receptor, the chimeric antigen receptor comprising anextracellular anti-CD19 binding moiety, wherein the anti-CD19 bindingmoiety comprises a humanized scFv sequence comprising a variable heavy(VH) domain of SEQ ID NO: 122, a hinge and/or transmembrane domain, anintracellular signaling domain, and wherein the polynucleotide alsoencodes membrane-bound interleukin-15 (mbIL15). In several embodiments,the polynucleotide encodes the humanized chimeric antigen receptor ofSEQ ID NO: 173, SEQ ID NO: 175, SEQ ID NO: 177, SEQ ID NO: 197, SEQ IDNO: 199, or SEQ ID NO: 201.

Provided for is a polynucleotide encoding a humanized CD19-directedchimeric antigen receptor, the chimeric antigen receptor comprising anextracellular anti-CD19 binding moiety, wherein the anti-CD19 bindingmoiety comprises a humanized scFv sequence comprising a variable heavy(VH) domain of SEQ ID NO: 123, a hinge and/or transmembrane domain, anintracellular signaling domain, and wherein the polynucleotide alsoencodes membrane-bound interleukin-15 (mbIL15). In several embodiments,the polynucleotide encodes the humanized chimeric antigen receptor ofSEQ ID NO: 179, SEQ ID NO: 181, SEQ ID NO: 183, SEQ ID NO: 203, SEQ IDNO: 205, or SEQ ID NO: 207.

In several embodiments, the provided for polynucleotides do not encodeSEQ ID NO: 112, 113, 114, or 116.

Provided for is a polynucleotide encoding a humanized CD19-directedchimeric antigen receptor, the chimeric antigen receptor comprising anextracellular anti-CD19 binding moiety; a hinge and/or transmembranedomain; an intracellular signaling domain, and wherein thepolynucleotide also encodes membrane-bound interleukin-15 (mbIL15), andwherein the polynucleotide is selected from the group consisting ofpolynucleotides having at least 95% identity to SEQ ID NO: 184, SEQ IDNO: 186, SEQ ID NO: 192, or SEQ ID NO: 200. In several embodiments, thepolynucleotide has the sequence of SEQ ID NO: 184, SEQ ID NO: 186, SEQID NO: 192, or SEQ ID NO: 200. In several embodiments, there areprovided engineered NK or T cells that express such a humanizedCD19-directed chimeric antigen receptor. Also provided for is a methodof treating cancer in a subject comprising administering to a subjecthaving cancer such engineered NK or T cells. Also provided is the use ofsuch polynucleotides in the treatment of cancer, such as in themanufacture of a medicament for the treatment of cancer.

EXAMPLES

The materials and methods disclosed herein are non-limiting examplesthat are employed according to certain embodiments disclosed herein.

According to several embodiments, NK cells are isolated from peripheralblood mononuclear cells and expanded through the use of a feeder cellline. As discussed in more detail below, in several embodiments, thefeeder cells are engineered to express certain stimulatory molecules(e.g. interleukins, CD3, 4-1 BBL, etc.) to promote immune cell expansionand activation. Engineered feeder cells are disclosed in, for example,International Patent Application PCT/SG2018/050138, which isincorporated in its entirety by reference herein. In severalembodiments, the stimulatory molecules, such as interleukin 12, 18,and/or 21 are separately added to the co-culture media, for example atdefined times and in particular amounts, to effect an enhanced expansionof a desired sub-population(s) of immune cells.

NK cells isolated from PBMC were cocultured with K562 cells expressingmembrane-bound IL15 and 4-1 BBL, with the media being supplemented withIL2. For one group of engineered NK cells, they were expanded in mediawas supplemented (at Day 0) with a combination of soluble IL12 andsoluble IL18. The media was refreshed with additional soluble IL12 andsoluble IL18 at Day 4. Additional details on embodiments of such culturemethodology is disclosed in U.S. Provisional Patent Application No.62/881,311, filed Jul. 31, 2019 and incorporated in its entirety byreference here. Viral transduction, with a CD19-directed chimericreceptor construct, was performed at Day 7. The resultant engineered NKcells were evaluated at 14, or more, days of total culture time.

Example 1

FIG. 5 depicts a schematic experimental model for evaluating theanti-tumor efficacy of engineered NK cells generated according tomethods disclosed herein. NOD-scid IL2Rgamma^(null) mice wereadministered 2×10⁵ Nalm6 cells (B cell precursor leukemia cell line)intravenously at Day 0. At Day 4, one group of mice received 2.7×10⁷ NKcells expressing an NK19 CAR (see FIG. 3A, though it shall beappreciated that other CD19-directed chimeric receptors could be used)while another group received NK cells that had been expanded usingsoluble IL12/1L18 (identified as NK19-IL12/18 cells). Leukemic tumorburden was assessed by fluorescent imaging performed at Days 3, 7, 11,18, and 25. FIG. 6A shows the imaging results from Days 3, 7, 11, and18. As can be seen, mice receiving either NK19 or NK19 IL12/18 hadsignificantly less tumor burden than mice receiving eithernon-transduced NK cells or PBS as a control. FIG. 6B shows a summary ofthe imaging data in a line graph (greater values of Flux(photons/second) indicates more fluorescent signal detection and greatertumor burden). Both NK19 and NK19 IL12/18 groups have less tumor burdenas early as Day 7 post-injection of the Nalm6 leukemia cells. Thisdifference is even more pronounced at Day 11, when the PBS and NT NKcell groups are exhibiting large amounts of tumor growth. Even as farout as Day 18, when tumor burden in the PBS and NT NK groups isextensive, the NK19 and NK19 IL12/18 groups show much less tumor burden.Unexpectedly, the NK19 IL12/18 groups show less tumor burden than thisreceiving the NK19 construct. This is surprising not only because NK19cells are quite efficacious at preventing leukemia cell growth, so thefurther enhancement of this effect is unexpected, but also because anupstream methodology of expanding the cells has not only impacted thecell number itself, but also the activity level of those expanded cells.

Example 2

Experiments were undertaken to determine if a given stimulatory domain(also referred to as co-stimulatory domains, given that many constructemploy multiple “signaling” domains in tandem, triplet or othermultiplex fashion) utilized in a CAR impacted expression (as well asactivity). NK cells were generated by transduction with viruses encodingvarious CARs depicted in FIGS. 3A-3C (though other constructs are used,in several embodiments). By way of non-limiting example, NK cells werecells were generated by transduction with a bicistronic virus encodingan anti-CD19 scFv, an intracellular OX40 costimulatory domain, CD3signaling domain, and membrane-bound IL-15 (see NK19, FIG. 3A) whichsupports prolonged cell survival and proliferation. The other CARconstructs tested, NK19-1, 2, 3, 4, 5, 8, 9, 10, 11, 12, and NK19-13,were transduced into NK cells in similar fashion. It shall beappreciated that, for those constructs that employ a Flag domain todetermine expression, analogous constructs not including the Flag (orother tag domain) are provided for, in several embodiments.

FIG. 7 summarizes the expression data of NK 19-1 to NK5 and NK8 toNK19-13 as evidenced by detection of CD19Flag. Data are presented aspercentage of NK cells expressing CD19-Flag relative to the total numberof NK cells presented. The data were collected at 4 days aftertransduction with the relevant virus encoding the NK19-“X” CAR. Asevidenced by the expression data, all constructs were expressed by atleast 55% of the NK cells. In fact, for eight of the eleven constructsfor which data was generated expression was detected in approximately75% or more of the total number of NK cells, with several constructsexpressed at over 80% efficiency. This expression data indicates that,according to several embodiments, selection of specific stimulatorydomains can enable a more efficient expression of the CAR by the NKcell. This is advantageous, in several embodiments, because a greaterportion of a given NK cell preparation is useful clinically (e.g., fewerinput NK cells needed to generate a clinically relevant engineered NKcell dose).

As discussed herein, various co-stimulatory domains can be employed inchimeric antigen receptors that Target CD19 (or other tumor markers).FIG. 8A depicts data related to the expression of CARs targeting CD19using various co-stimulatory domains. By way of non-limiting example,co-stimulatory domains can include, but are not limited to, OX40, CD28,iCOS, CD28/41 BB, CS27, and CD44). FIG. 8A shows mean fluorescenceintensity data representing expression of the indicated CAR constructsby NK cells. As evidenced by the low MFI detected for the GFP control,these data indicate that these construct (a) are expressed by NK cells,and (b) are expressed relatively stably by NK cells over a 4 week periodpost-transduction. FIG. 8B shows the efficiency of expression of theCARs with the indicated co-stimulatory domains. While there is somevariability in efficiency of expression, ranging from about 60% to about80% efficiency, each of the CARs with the indicated co-stimulatorydomains expressed well, and also expressed relatively consistently overat least 4 weeks.

With respect to the cytotoxic efficacy of the NK cells expressing theCD19-directed CARs utilizing the various co-stimulatory domains, thatdata is shown in FIGS. 9A-9F. Cultured Nalm6 or Raji cells were exposedto engineered NK cells expressing the indicated NK19 constructs andco-cultured for the indicated number of days (X axis represents days) ofexposure to NK19-X-expressing NK cells. FIG. 9A shows the cytotoxiceffects of the indicated constructs against Nalm6 cells 7 days after NKcells from a first donor were transduced with the indicated construct.The effector cell to target cell ratio for this experiment was 1:1. Asshown in the traces, each of NK19-10, NK19-8, NK-1911, NK19-5, andNK19-12 allowed increases in the number of Nalm6 cells detected, on parwith that of NK cells expressing only GFP as a control. However, each ofNK19-3, NK19-9, NK19-4, NK19-13, NK19-1 and NK19-2 showed significantlyless increase in Nalm6 cell number (e.g., greater cytotoxicity). Inseveral embodiments, such constructs are therefore expressed in NK cellsand used in treating B cell leukemia (or other tumor types). In severalembodiments, one or more of the stimulatory domains from one of theconstructs is engineered into another construct with a differentstimulatory domain, which advantageously results in synergisticsignaling and further enhanced cytotoxicity. By way of non-limitingexample, an NK19-1 construct with an OX40 stimulatory domain is, inseveral embodiments, further engineered to also express a CD44stimulatory domain in addition to OX40. By way of further non-limitingexample, an NK19-1 construct with an OX40 stimulatory domain is, inseveral embodiments, further engineered to also express a CD44stimulatory domain and a CD17 stimulatory domain in addition to OX40.

FIG. 9B shows corresponding cytotoxicity data for engineered NK cellsfrom a second donor against Raji B-cell leukemia cells, 7 dayspost-transduction. The effector cell to target cell ratio here was also1:1. As shown, similar to the engineered constructs activity againstNalm6 cells, several constructs allowed for increased Raji cell count.However, each of NK19-13, NK 19-4, NK19-2, NK19-3, NK19-1, and NK19-9prevented Raji cell growth to a substantial degree, with several of theconstructs resulting in nearly no Raji cell growth. In severalembodiments, such constructs are therefore expressed in NK cells andused in treating B cell leukemia (or other tumor types).

FIG. 9C shows data for NK cells from Donor 1 against Nalm6 cells 14 dayspost-transduction. The effector to target cell ratio is 1:1. As shown,even at two weeks post-transduction, the NK19-13, NK19-4, NK19-3,NK19-2, NK19-1, and NK19-9 expressing NK cells prevented virtually allNalm6 cell growth, indicative of their highly cytotoxic effect againsttumor cells. In several embodiments, such constructs are thereforeexpressed in NK cells and used in treating B cell leukemia (or othertumor types). As discussed above, in several embodiments, a construct isgenerated that employs a combination two, three, or more of thestimulatory domains, resulting in a synergistic NK cell stimulation andenhanced cytotoxicity.

FIG. 9D shows data for NK cells from Donor 2 against Raji cells at 14days post-transduction. The effector cell to target cell ratio was 1:2.As shown, NK cells expressing GFP only allowed growth of Raji cellsessentially the same as untreated Raji cells. In contrast, each ofNK19-3, NK19-1, NK19-4, NK19-13, NK19-2, and NK19-9 expressing NK cellssignificantly retarded the growth of Raji cells, with several of theconstructs allowing little to no Raji cell growth. In severalembodiments, such constructs are therefore expressed in NK cells andused in treating B cell leukemia (or other tumor types). As discussedabove, in several embodiments, a construct is generated that employs acombination two, three, or more of the stimulatory domains, resulting ina synergistic NK cell stimulation and enhanced cytotoxicity.

FIG. 9E shows summary data for cytotoxicity against Nalm6 cells at anE:T ratio of 1:1 and 1:2 at 7 days post-transduction. At an E:T ratio of1:2, all but one of the NK19 constructs was equivalent to, or more,cytotoxic than NK cells expressing GFP alone. In fact, even while at a1:2 ratio, six of the constructs achieved ˜50% or greater cytotoxicity.When tested at an E:T of 1:1, seven of the constructs achieved ˜50% orgreater cytotoxicity, however, five of the constructs (NK19-13, NK19-2,NK19-9, NK19-3, and NK19-1) yielded cytotoxicity exceeding 70%. FIG. 9Fshows the corresponding data for Raji cells. All constructs testedshowed enhanced cytotoxicity over GFP-expressing NK cells at both 1:2and 1:1 E:T ratios. At an E:T of 1:2, four of the constructs exceeded40% cytotoxicity, while at 1:1, seven constructs exceeded that killrate. Furthermore, at a 1:1 E:T, three constructs yielded cytotoxicityof 60% or more, with the most efficacious construct achieving nearly 90%cytotoxicity. Taken together, these data demonstrated that variousCD19-directed CAR constructs can not only be expressed, but are stablyexpressed, and are also effective an inducing cytotoxicity in multiplecancer cell types, in some cases exceeding an 80% kill rate. Accordingto additional embodiments, CD19-targeting constructs are generated thatemploy combinations of two, three or more stimulatory domains, whichresult in further enhancements in the cytotoxicity of the NK cellsexpressing them. In several embodiments, CD19-directed constructs cansynergistically interact with NK cells expressing receptors directedagainst other tumor markers, such as ligands of NKG2D (such chimericreceptor bearing NK cells are described in PCT/US2018/024650, which isincorporated by reference herein in its entirety). For example, achimeric receptor comprising an binding domain that binds ligands ofNKG2D, an OX40 stimulatory domain, and a CD3zeta signaling domain couldbe used in conjunction with any of the CD19-targeting constructsdisclosed herein. In several embodiments, such a chimeric receptor is atleast 90% identical in sequence to the nucleic acid sequence of SEQ IDNO: 145. In several embodiments, such a chimeric receptor is at least90% identical in sequence to the amino acid sequence of SEQ ID NO: 146.

Further experiments were performed to evaluate the cytotoxicity ofselected CD19-directed CAR constructs. NK cells isolated from threedifferent donors (two donors for 14 day experiment) were transduced withvectors encoding the indicated constructs, NK19-1 (OX40 co-stimulatorydomain); NK19-2 (CD28 co-stimulatory domain); NK19-3 (ICOSco-stimulatory domain); NK19-4 (CD28-41BB co-stimulatory domain); NK19-9(CD27 co-stimulatory domain); and NK19-13 (CD44 co-stimulatory domain).Seven (n=3) or 14 (n=2) days after transduction, those engineered NKcells were co-cultured with Nalm6 or Raji cells at an E:T ratio of 1:1.Results are shown in FIG. 10A (expressed as percent enhancedcytotoxicity over NK cells expressing GFP). Consistent with the datafrom FIG. 9, each of the constructs tested yielded enhanced cytotoxiceffects against Nalm6 cells, ranging from a mean 40% increase withNK19-4 to an overall average of about 50% increase with the other 5constructs. FIG. 10B shows the corresponding data against Raji cells.Again, each construct outperformed NK cells expressing only GFP, withmean increases in cytotoxicity over GFP NK cells ranging from about 40%increase to about 50% increase. Using cells from two donors at 14 dayspost-transduction, NK19-1, NK19-9 and NK19-13 were tested on Nalm6 andRaji cells. FIG. 10C shows the calculated enhanced cytotoxicity of theseconstructs over GFP-expressing NK cells, where average increases ofnearly 80% were seen with NK19-9 expressing NK cells, about 75% withNK19-1 expressing cells and over 60% with NK19-13 expressing cells.Similar results were obtained against Raji cells—NK19-13 expressingcells showed over a 20% improvement in cytotoxicity, NK19-1 expressingcells exhibiting nearly 60% enhanced activity and NK19-9 expressingcells showing almost 70% more cytotoxic activity against Raji cells.These results further support the embodiments disclosed herein whereinengineered NK cells expressing CD19-directed CARs are provided, as aremethods for their use in treating cancer immunotherapy results inenhanced cytotoxicity against target tumor cells.

FIGS. 11A-11E depict data related to the cytokine release profiles fromNK cells when cultured the Nalm6 cells, which ties into the mechanism bywhich NK cells control tumor and virus-infected cells—through releasingcytotoxic granules and proinflammatory cytokines. FIG. 11A shows that ascompared to control, or even GFP-expressing NK cells, NK cellsexpressing NK19-1, NK19-9, or NK19-13 all express greater concentrationsof the serine protease, Granzyme B, that is present in the granulesreleased by NK cells. NK cells expressing these CD19-directed CARsreleased approximately 4 times more Granzyme B than controlGFP-expressing NK cells. FIG. 11B shows data related to increasedrelease of perforin by the engineered NK cells. Interestingly, perforinlevels were not substantially elevated over the concentrations resultingfrom GFP-expressing NK cells (though perforin concentration was elevatedover control. Perforins work in concert with Granzyme B (and othergranzymes), with perforins functioning to generate pores through a cellmembrane to allow granzymes to cross the membrane, then exert theirprotease effects on intracellular protein targets. The data raise thepossibility that the perforin release while approximately the same, orreduced in connection with certain constructs, are actually moreefficient at pore-formation, thus allowing the same degree of poreformation. Alternatively, if the perforins released from NK19 expressingNK cells are no more efficient at pore formation, this is offset by theelevated increase of granzyme B (and/or other granzymes). Thus, enhancedcytotoxicity is still achieved.

FIG. 11C shows that as compared to control, or even GFP-expressing NKcells, NK cells expressing NK19-1, NK19-9, or NK19-13 all releasegreater concentrations of the inflammatory cytokine TNF alpha. FIG. 11Dshows similar data for GM-CSF release by NK19-expressing NK cells andFIG. 11E shows similar data for interferon gamma release. Likewise, whentested on Raji cells, similar patterns of release result, as shown inFIGS. 12A-12E. These data indicate that the NK19-epressing cells exerttheir cytotoxic effects, at least in part, through the increased releaseof inflammatory cytokines and/or cytotoxic granules. As mentioned above,in several embodiments, the engineered CARs are designed to havecombinations of two, three or more co-stimulatory domains, withsynergistically increased cytokine/granule release and cytotoxicityagainst target cancers.

Further evidencing the enhanced effects of NK19 constructs on tumorprogression, NSG mice were injected on Day 0 with 1×10⁵ Nalm6 cells(expressing a fluorescent reporter) intravenously. At Day 3, micereceived either a PBS control injection, non-transduced NK cells(“NTNK”, 10M), NK19-2 expressing NK cells (10 million cells), NK19-9expressing cells (10 million), NK19-1 expressing cells (10 million, orNK19-1 expressing cells (30 million). Fluorescent imaging to detect theNalm6 cells was performed on Days 3, 8, 11, 18, and 25 (imaging data notshown). The data is shown in FIG. 13. As shown, the injection of NKcells expressing any NK19 variant resulted in a reduced progression ofNalm6 growth. NK19-2 expressing NK cells showed minor Nalm6 growth onDay 8, with more Nalm6 growth by Day 11, and substantial growth by Day18 (though less than with NTNK cells). Neither NK19-9 or NK19-1 (ateither dose) expressing NK cells showed detectable tumor burden on Day 8by imaging. Mice receiving NK19-9 expressing NK cells did show someincrease in Nalm6 cell growth by Day 11, with further progression by Day18. At Day 11, mice receiving either dose of NK19-1 expressing cells didnot exhibit Nalm6 cell growth. By day 18, mice receiving 10 millionNK19-9 cells showed some tumor growth. However, with a 30 million celldose, those mice receiving NK19-9 expressing NK cells showed only minoramounts of Nalm6 cell growth.

FIG. 14A depicts a line graph of the bioluminescence intensity showndetected in the mice (e.g., the fluorescent signal shown in FIG. 13,note that FIG. 13 does not show imaging data for Day 25). Consistentwith the images of FIG. 13, the line graph of FIG. 14A shows increasedNalm6 cell count (as represented by increased BLI) for all groups at Day25, with significant cell numbers detected in PBS and NTNK groups, andsomewhat less for NK19-2, NK19-1 (10M) and NK19-9 groups. NK19-1 (30M)showed the smallest increase, representing that construct's ability toreduce the rate of Nalm6 progression due to cytotoxic effects on theNalm6 cells. While each construct eventually allows some Nalm6 growth,even if modest, the tested NK19 constructs delayed the onset of thatgrowth, as evidenced by the flat line through Day 11 for the NK19curves. To better show that aspect, the data are replotted on a logscale Y-axis in FIG. 14B, which allows for curve separation. Asdisplayed, the NTNK and PBS curves show an upward trend after Day 3,indicating nearly immediate Nalm6 growth. In contrast, the NK19-9,NK19-2 and both NK19-1 curves either drop, or only slightly trend upwardthrough Day 7. At Day 11, consistent with the images in FIG. 13, Nalm6cell growth is detected in the NK19-9, NK19-2 and NK19-1 (10M) groups.In contrast, the NK19-1 (30M) group is still approximately at baseline,reflecting the lack on any significant Nalm6 cell growth. Cell growthtrends upward in the NK19-9 (30M group) on Day 18. While increases intumor cell number occur, even with the NK19 constructs beingadministered, the delay of the growth onset could be advantageous inseveral embodiments. For example, this presents an opportunity tore-dose a patient with another dose of engineered NK cells expressing aCD19-directed construct. In several embodiments, a subsequent dose (ascould the initial dose) may optionally comprise NK cells that have beenedited to reduce allogenicty, for example by gene editing. Thus, inseveral embodiments, two, three, four or more doses of CD19-directed CARexpressing NK cells are administered. This delay in tumor cell growthpresents an opportunity to dose, either serially (or concurrently) withan NK cell expressing a chimeric construct directed to a different tumormarker and/or some other variety of anti-cancer therapy (e.g.,checkpoint inhibitor, antibody therapy, chemotherapy, etc.). FIG. 14Cdepicts data related to CD3 expression of cells transduced with thevarious constructs within blood samples taken from mice treated asindicated in the X axis. CD3 is a T cell marker. As indicated, but for Tcells engineered to express the NK19-1 construct and a mixed populationof NK cells and T cells, CD3 expression was essentially negligible. FIG.14D depicts data related to CD56 expression, which is a marker for humanNK cells. While there is some small amount of background staining, theblood samples from mice treated with NK cells expressing the indicatedconstructs is relatively low (as would be expected given that (i) theblood is a murine blood sample and murine cells would make the majorityof the total, and (ii) CD56 is detecting only human NK cells (e.g.,those administered). The data are consisting in that regard, with the30M NK19-1 treatment group shoring markedly more CD56 expression thanthe other groups, and T cells/NK+ T cells groups expressing CD56 atbackground levels. FIG. 14E shows data related to the GFP expression bytumor cells. The blood samples were collected at −3 weeks into the invivo experiment. As shown (consistent with the images/BLI data), thecontrol PBS and NTNK groups exhibit higher percentages of GFP expression(e.g., a greater percentage of the total of live blood cells in thesample is tumor cells). Each of the engineered constructs showssignificantly less GFP expression, based on the engineered constructscontrolling/reducing tumor cell growth. FIG. 14F shows similar data toFIG. 14D, but measures CD19 expression across all the live cells in agiven murine blood sample. As with GFP, the PBS and NKNT groups showCD19 expression at approximately 10-12%, meaning 10-12% of the livecells in the blood sample are tumor cells, the remainder being murineblood cells. As shown in the other experimental group, CD19 expressionis much lower, reflective of the engineered CAR constructs limiting thegrowth of the tumor cells. These data are in accordance with embodimentsdisclosed herein, wherein engineered NK cells expressing CD19-directedCARs are highly cytotoxic and allow for the treatment of canceroustumors.

Further Evaluation of Humanized Constructs

As discussed in detail above, in several embodiments, severalembodiments of the CARs disclosed herein involve the use of humanizedsequences, such as in the extracellular binding moiety. In severalembodiments, one or more aspects of that region is subjected to ahumanization campaign. In several embodiments, one or more of the heavyand/or light chain of an antibody is humanized, which (as discussedabove) can provide advantages including, but not limited to, reducedimmunogenicity, increased stability, longer efficacy, increased potency,and the like.

Example 3

FIG. 15 shows a schematic of a series of humanized constructs accordingto several embodiments disclosed herein. Such constructs are designatedby “H” in their identifier. By way of explanation, NK19H-1 is ahumanized anti-CD19 CAR employing an scFv made up of a first light chainand a first heavy chain CU H1′), while NK19H-3 employs an scFv made upof a third light chain and the first heavy chain (‘L3H1’). FIG. 15 alsoshows data related to the stability and aggregation of the variouscombinations of heavy and light chains following transient expressionand secretion from 293T cells. Data are shown related to the meanfluorescence intensity detected by flow cytometry when each antibody washeated to 70° C. and then cooled back to room temperature (‘Heating’)vs. the same variant held on ice (‘Unheating’). After heat treatment,the ScFv variants are used in a flow cytometry protocol at variousconcentrations (0.4, 0.25, and 0.125 ug/mL). Loss of fluorescenceintensity indicates that the ScFv either lost structural integrity oraggregated through the heating process. ScFv's with the best thermalstability as indicated by comparable MFI under both conditions arefavored for further development, according to some embodiments.

After having assessed the stability of the constructs, selectedanti-CD19 CAR constructs were further evaluated. FIG. 16 shows summarydata of expression of the indicated constructs by the NK cells of 3donors (#140, #9, and #20). As depicted in the Figure, expression isevaluated by detection of CD19Flag. Data are presented as percentage ofNK cells expressing CD19-Flag relative to the total number of NK cellspresented. The data were collected at 4 days after transduction with therelevant virus encoding the NK19H-“X” CAR. As evidenced by theexpression data, each of the selected constructs were expressed by NKcells, to varying degrees. Expression levels ranged from expression byabout 20% of the total NK cells with NK19H-2, NK19H-11 and NK19H-12.Most of the other constructs were successfully expression by ˜40%-60% ofthe NK cells, which is on par with expression of the non-humanizedNK19-1 construct. The NK19H-5 construct was expressed by over 80% of theNK cells. While certain constructs may be more efficiently expressed,those with lower expression levels were still evaluated because, as withseveral embodiments such constructs still exhibit significantcytotoxicity against target cells. According to several embodiments,however, those with higher expression efficiency can be advantageous,for example because a greater portion of a given NK cell preparation isuseful clinically (e.g., fewer input NK cells needed to generate aclinically relevant engineered NK cell dose).

FIGS. 17A-17E depict such cytotoxicity data. FIG. 17A shows cytotoxicityof NK cells from Donor 20 and transduced with the indicated constructsagainst the CD19-positive Nalm6 leukemia cell line (at an E:T of 1:1;20K cells per well). The data shows that, despite the varied expressionlevels, most of the NK19H constructs were able to exert cytotoxiceffects against the Nalm6 target cells. NK19H-11 showed the leastefficacy, allowing Nalm6 cell growth at levels just below the controls.In contrast, NK19H-1 and NK19H-3 showed cytotoxicity on par with thenon-humanized NK19-1 construct, allowing for some cell growth at thelater time points of co-culture. Notably NK19H-4 and NK19H-5 showedsignificant cytotoxicity, allowing only very limited Nalm6 growththroughout the experiment.

FIG. 17B shows NK cells from Donor 20 tested against the CD19-positiveBurkitt's lymphoma cell line Raji. As with Nalm6 cells, the varioushumanized anti-CD19 constructs showed variable cytotoxicity against thetarget cells. Similar to the data of FIG. 17A, the NK19H-11 constructshowed limited cytotoxicity, but all other constructs showed promisingcytotoxicity against the target cells, with NK19H-1 performing on parwith the non-humanized NK19-1 construct, and each of NK19H-3, 19H-4, and19H-5 constructs inducing greater levels of cytotoxicity, limiting Rajigrowth until the later stages of the co-culture (with NK19H-5 allowingonly very limited Raji cell growth). FIG. 17C shows corresponding Nalm6data from Donor 140. Here, a similar pattern of efficacy was detected,with NK19H-11 allowing Nalm6 growth approximating negative controls.However, each of NK19H-1, 19H-3 and 19H-4 induced at least as muchcytotoxicity as non-humanized NK19-1. Again, NK19H-5 showed significantcytotoxicity, limiting Nalm6 growth throughout the experiment. FIG. 17Dshows data from Donor 9 against Raji cells. Only NK19H-11 allowed anysubstantial Raji cell growth. In contrast, NK cells from this donorexpressed the non-humanized NK19-1 or any of the humanized NK19H-1,19H-3, 19H-4, or 19H-5 constructs suppressed any Raji cell growththrough the induced cytotoxic effects. FIG. 17E shows data for NK cellsfrom Donor 9 against Nalm6 cells. In this experiment, the cytotoxicityof three of the humanized constructs (NK19H-11, 19H-1, and 19H-3) waslimited. There is some donor to donor variability, as certain of theseconstructs induced cytotoxicity when expressed by NK cells of otherdonors. The NK19H-4 and 19H-5 constructs exhibited significant cytotoxiceffects, nearly limiting Nalm6 growth to zero over the course of theexperiment. Taken together, these data complement the expression dataand show that, in accordance with several embodiments, humanized CARconstructs targeting CD19 are effective at killing tumor cells, even ifthey have variable expression efficiencies. Additionally, according tosome embodiments, the expression efficiency is not correlated withcytotoxicity and even constructs with limited expression efficiency candemonstrate significant cytotoxicity.

Example 4

Further experiments paralleling those discussed above were performedusing NK cells from additional donors. FIG. 18 shows constructexpression efficiency for three donors (#945, 137 and 138) for theindicated constructs. As with the prior experiment the data presentedrepresent the number of CD19-Flag positive cells out of the total numberof NK cells evaluated. The data for these donors shows a higher overallefficiency of expression for all but one of the humanized constructs.Only NK19H-3 was expressed less than the non-humanized NK19-1 construct.With these donor NK cells, expression of the humanized constructs wasdetected on about 70% to 80% of the NK cells. As above, further toevaluation of expression, cytotoxicity against CD19 expression targetcells was evaluated. FIG. 19A shows data related to NK cells from adonor (#137) expressing the indicated constructs and co-cultured withRaji cells. In this experiment, the engineered NK cells expressing theindicated constructs were exposed to the tumor cells at two time points,7-days and an additional bolus of tumor cells was added at 14-dayspost-transduction. The arrow shows the second administration of tumorcells. As shown, untreated Raji cells expanded throughout theexperiment. NK cells expressing GFP or non-humanized NK19-1 induced somecytotoxicity as shown by the reduced Raji cell growth as compared tocontrol. NK cells expressing NKH19-3 (the humanized construct with thelowest expression efficiency) were also able to reduce Raji growth. Eachof the other humanized NK constructs were able to reduce Raji cellgrowth compared to controls, even at 14 days post-transduction, which isindicative of the enhanced persistence of engineered NK cells disclosedherein. FIG. 19B shows corresponding data for Donor 137 NK cells againstNalm6 cells with engineered NK cells again being added at day 7 (day 0of experiment) and day 14 post-transduction (˜day 7 of experiment).Cytotoxicity of the indicated constructs was more variable in thisparticular experiment. However, several humanized anti-CD19 constructswere able to produce marked cytotoxicity and reduce Nalm6 cell growth ascompared to controls. These data suggest that, according to severalembodiments, a more frequent dosing schedule (e.g., every 2 days, every3 days, every 4 days, every 5 days, etc.) would be beneficial forcertain subjects. Advantageously, in several embodiments, engineered NKcells as disclosed herein are allogeneic and can be readily used in morefrequent dosing regimens. FIG. 19C shows corresponding data for Donor138 against Raji cells. Here, many of the humanized constructs still hadsignificant cytotoxic effects on the Raji cells, even with the seconddose of Raji cells at 14 days post-transduction. Six of the 7 humanizedconstructs showed this behavior, and outperformed the non-humanizedNK19-1 construct. FIG. 19D shows the corresponding data for Donor 138against Nalm6 cells. While the additional bolus of Nalm6 led toincreased Nalm6 growth in the latter stages the experiment, nearly allof the humanized constructs performed better than controls. In fact,NK19H-5-bearing NK cells were able to limit Nalm6 growth until the finalfew days of the experiment (after the second dose). As above, these datashow that humanized anti-CD19 CAR constructs can not only be expressedby NK cells, but can also exert cytotoxic activity against target cells,with an enhanced persistence. In several embodiments, this allowsmultiple dosing to be separated by longer periods of time, which couldbe advantageous for treating cancers, while limiting potentialimmunogenicity (at least in part due to the humanization and/or becauseof the reduced frequency of administration).

Example 5

Further data for various humanized constructs in additional donors wascollected. FIGS. 20A-20B show expression data for various anti-CD19 CARconstructs in NK cells from two additional donors. FIG. 20A shows themean fluorescence intensity for NK cells at 10 days post-transduction.As indicated, as in accordance with several embodiments disclosedherein, each of the humanized anti-CD19 CAR constructs showed enhancedoverall expression as compared to a non-humanized anti-CD19 CAR. FIG.20B shows the expression data of CD19-Flag positive NK cells as apercentage of the total number of NK cells analyzed. As shown, each ofthe humanized anti-CD19 constructs were more efficiently expressed thanthe non-humanized construct (which was already expressed by almost 80%of the NK cells). The humanized CARs were expressed by approximately 85%to 95% of the NK cells, depending on the construct. FIG. 21A showscytotoxicity data for engineered NK cells from Donor 703 (one of the twodonors from FIG. 20) against Raji cells co-cultured with the NK cells atday 7 post-transduction and again at day 14 post-transduction. As shownin the Figure, Raji cells and NK cells expressing GFP alone grewrobustly through day 7 and again through day 14. The non-humanizedanti-CD19 CAR NK19-1 was able to suppress Raji cell growth through 7days, and allowed relatively limited growth through 14 days. Each of thehumanized anti-CD19 CAR constructs further suppressed Raji cell growthwith several of the constructs nearly completely suppressing Raji cellgrowth. FIG. 21B shows corresponding data for cytotoxicity against Rajicells for NK cells isolated from Donor 877. These data show a similartrend to that from the prior donor. The two control groups allowedsignificant Raji cell growth, while each of the humanized anti-CD19 CARconstructs yielded significant cytotoxic effects.

FIGS. 22A-22E show cytokine release profiles from Raji cells co-culturedwith NK cells expressing the indicated constructs. FIG. 22A shows IFNgrelease by NK cells co-cultured with Raji cells. Each of the indicatedhumanized constructs resulted in increased IFNg release as compared tothe non-humanized NK19-1 construct. Similarly, as shown in FIG. 22B, thehumanized anti-CD19 constructs enabled the NK cells to release greateramounts of GM-CSF as compared to control NK cells. FIG. 22C show thathumanized anti-CD19 CAR constructs induce elevated TNF release from theNK cells. Perforin levels were not significantly different across theconstructs tested, as shown in FIG. 22D. Likewise Granzyme levels wererelatively constant across the constructs. These data, taken together,indicate that according some embodiments, humanized anti-CD19 CARconstructs expressed on NK cells cause those NK cells to release greateramounts of one or more cytokines that lead to cytotoxic effects ontarget cells.

Example 6

While the data presented above show that humanized anti-CD19 CARs can beexpressed by NK cells and have cytotoxic effects on target tumor cells,additional experiments were performed to determine the longevity of theengineered NK cells. NK cells expressing the indicated anti-CD19 CARconstructs were cultured and the cell count was measured at various timepoints, out to 26 days post-transduction. FIG. 23A shows the survivaldata for NK cells from Donor 137. The data for the GFP-expressing NKcells show that the cell count at day 7 is higher than the cell count atany other time-point, suggesting that GFP has provided no additionallongevity-inducing effects to the NK cells. Likewise, NK cellsexpressing non-humanized NK19-1 shows a fall off of cell number overtime. While each of the humanized constructs shows some variability interms of cell count, the trend of the data shows that expression of thehumanized anti-CD19 constructs results in less NK cell death over time.A similar trend is shown in the data of FIG. 23B, which shows cellviability for NK cells from Donor 138. While the timing of analysis ismodified, FIG. 23C shows data yielding a similar trend (for Donor 703),in that the expression of the humanized constructs results in longer NKcell survival over time in culture. FIG. 23D shows data for NK cellsfrom Donor 877, where the expression of humanized anti-CD19 CARconstructs allows for reduced NK cell death over time in culture.

Summary data for the expression of the various anti-CD19 CAR constructsis shows in FIG. 24, which displays the expression efficiency of theindicated constructs at day 11 post-transduction. As anticipated, theGFP-transduced NK cells exhibit no CD19-Flag expression, serving as anegative control. Likewise, the non-humanized NK19-1 construct serves asa positive control. Each of the humanized constructs assessed showedenhanced expression efficiency as compared to NK19-1, with efficienciesranging from about 85% to about 95% (e.g., 85%-95% of all the NK cellstested expressed the Flag-tagged CAR construct).

FIGS. 25A-25I show raw flow cytometry data for one donor whereinexpression of CD19-Flag (indicative of CAR expression) is measured. FIG.25A shows the GFP-control, with little to no Flag expression. FIG. 25Bshows Flag detection with the NK19-1 positive control. FIGS. 25C-25Ishow the results of Flag detection for the indicated humanized anti-CD19CAR constructs, with the percentage of cells expressing the constructsranging from about 80% to about 95%. As with the experiments above,these data confirm that the humanized anti-CD19 CAR constructs can berobustly expressed by transduced NK cells.

Example 7

Similar to the experiments discussed above, Raji cells were exposed toNK cells from donor 103 which were transduced with the variousconstructs indicated (see FIG. 26A). Raji cells were co-cultured on day7 post-transduction of the NK cells, and the NK cells were re-challengedagain on day 14. As expected Raji cells alone exhibited continuedgrowth, while GFP-transduced NK cells reduced that cell growth a smallamount. In contrast, the positive control non-humanized NK19-1 constructreduced Raji cell growth until the very late stages of the experiment.Each of the humanized anti-CD19 CAR constructs yielded enhancedcytotoxicity against the Raji cells. Three of the constructs (NK19H-2,H-3, and H-4) allowed for minor Raji cell growth at the late stage ofthe experiment, while the other humanized constructs effectivelyeliminated Raji cell growth, even with the re-challenge at day 14. FIG.26B shows corresponding data regarding the cytotoxicity of donor 103 NKcells against Nalm6 cells. As indicated in the Figure, all of theanti-CD19 CAR constructs (whether humanized or not) appeared exhibitsignificant cytotoxicity against the Nalm6 cells through 7 days ofco-culture. However, Nalm6 growth increased for all groups with the day14 re-challenge. While the growth curves initially were somewhat flat,Nlam6 growth later increased in rate. These data suggest that, accordingto some embodiments, a more frequent dosing strategy is employed to keeptarget cells from reaching a threshold growth rate. In severalembodiments, a larger dose is given and/or a dose is given morefrequently, to prevent target cell growth under a physiologicalequivalent context to a “re-challenge.”

FIG. 26C shows data for Raji cells (as with FIG. 26A) with NK cells fromdonor 275. Similar to those of FIG. 26A, each of the anti-CD19 CARconstructs showed significant cytotoxicity against Raji cells, andallowing limited growth of the target cells, even with a re-challenge.FIG. 26D shows data for Nalm6 cells (as with FIG. 26B) with NK cellsfrom donor 275. Results for this experiment were also similar to thosefor donor 103, with effective control of Nalm6 cells through 7 days, butreduced cytotoxicity after re-challenge. These data indicated thatdosing strategies, depending on the embodiment, are developed for aspecific donor and/or for a specific target tumor type. For example,while Nalm6 cells are CD19 positive, they may express less CD19 than,for example, Raji cells, thereby accounting for the reduced cytotoxicityof NK cells from donor 103 and 275 against the Nalm6 cell line. Theefficacy of the transduced NK cells against the Raji cells indicatesthat the NK cells are capable of cytotoxicity, even showing persistenceout to nearly two weeks post-transduction. Thus, according to severalembodiments, an NK cell dose and/or dosing frequency can be tailored toa given patient's tumor type, native NK cell activity, and/or theaggressiveness/stage of a cancer to allow robust and ongoingcytotoxicity against the target cells and achieving control of tumorburden.

FIGS. 27A-27B show flow cytometry data that characterizes NK cells fromtwo donors (276 in FIG. 17A and 877 in 27B) transduced with non-limitingexamples of anti-CD19 CAR constructs as disclosed herein. FIGS. 27A and27B both show that NK cells transduced with GFP express little to noCD19-Flag. Each of the other panels demonstrates that the NK cells fromthese donors can express not only the positive control non-humanizedNK19-1 construct, but also efficiently express the selected humanizedanti-CD19 CAR constructs. This is indicative of the limited impact thathumanization of the anti-CD19 binder has on expression characteristics.

FIGS. 28A-28B show initial data for NK cell cytotoxicity of those twodonors from FIG. 27 against Raji cells at an E:T ratio of 1:1.Consistent with results discussed above, each of the selected humanizedanti-CD19 CAR constructs endowed transduced NK cells with significantcytotoxic potential against the target Raji cells. Each of the indicatednon-limiting examples of anti-CD19 CAR constructs effectively controlledRaji cell growth (equivalent to, or enhanced as compared to, thenon-humanized NK19-1 construct) with the trend of decreasing Raji cellnumbers even as long as 70 hours after inception of the experiment.Similarly, FIG. 28B shows that transduced NK cells from donor 877 alsoeffectively control Raji cell growth, equivalent to, or enhanced ascompared to, the non-humanized NK19-1 construct. These data are in linewith those presented for NK cells from other donors, discussed above,and indicate that, in accordance with several embodiments disclosedherein, transduction of NK cells with humanized anti-CD19 CARs enablethe engineered NK cells to exert significant cytotoxic effects againsttarget tumor cells and can effectively control growth of tumor cells.According to several embodiments, such engineered anti-CD19 NK cells(whether allogeneic or autologous) allow for robust and effectivecellular immunotherapy to treat cancers.

Example 8

Further experiments were conducted to evaluate humanized CD19 CARconstructs as disclosed herein. FIG. 29A shows a schematic of theexperimental protocol. Briefly, NSG mice were injected on Day 0 with1×10⁵ Nalm6 cells (expressing a fluorescent reporter) intravenously. AtDay 1, mice received either a PBS control injection, non-transduced Kcells (“NTNK”, 10M), 10 million NK cells expressing the non-humanizedNK19-1 CAR, or 10 million NK cells expressing one of various humanizedCD19 CAR constructs. Blood collection and fluorescent imaging wereperformed as indicated. Bioluminescence data is shown in FIG. 29B. Asshown, the injection of NK cells expressing any CD19 CAR constructresulted in a reduced progression of Nalm6 growth. The humanizedconstructs tested (NK19-H2, NK19-H5, and NK19H-9) each showedsignificant delay in Nalm6 growth compared to controls. FIG. 29C is aline graph depicting the measured bioluminescence over 31 days. Asshown, each of the humanized constructs tested in this experiment showedreduced tumor progression as compared to controls, and slightly reducedas compared to non-humanized NK19-1. FIG. 29D shows a survival curvethat reflects the reduction in tumor progression, with the mice treatedwith NK19-1 or any of the selected humanized constructs surviving longerthan the control groups. FIG. 29E shows data related to an evaluation ofthe expression of each of the humanized constructs on the NK cells. Asshown, each of the humanized constructs expressed more robustly than thenon-humanized NK19-1 construct. In accordance with several embodimentsdisclosed herein, the use of a humanized construct results in a moreefficacious therapeutic, at least in part due to the enhanced expression(and/or activity) of the CAR by the NK cells.

FIGS. 30A-30F relate to characteristics of cells in the blood of themice treated with the indicated constructs at various time points duringthe experiment. FIG. 30A shows the percentage of human CD56+ cells inthe peripheral blood of the mice, which represents the survival of theadministered NK cells through the first fifteen days of the experiment.Each of the experimental groups exhibit a higher percentage of cells,with the NK19-H2 construct exhibiting the most persistence of thosetested. FIG. 30B shows similar data, based on the detection of the flagexpression tag used in these constructs (though, in several embodiments,no tag is used). These data show that the humanized constructs areexpressed at greater levels than the non-humanized constructs. FIG. 30Cshows the detection of GFP positive tumor cells after 15 days. TheCAR-expressing NK cells show nearly zero GFP expression, reflective oftheir inhibition of Nalm6 growth at Day 15. FIGS. 30D-30F show similardata at Day 32. These data show that the NK cells expressing humanizedCD19 CAR constructs make up a greater percentage of the cells present inthe peripheral blood of the mice tested, which is consistent with theincreased efficacy of these constructs at controlling tumor growth atlater time points. FIG. 30F shows that there is little difference inexpression among the humanized CD19 CAR constructs at day 32. FIG. 32Freflects the enhanced ability of NK cells expressing the humanized CD19CAR constructs at controlling tumor growth, with the detectedGFP-positive cells being lower in the humanized treatment groups, evenas compared to the animals receiving NK19-1. In several embodiments, theenhanced efficacy is due, at least in part, to the enhanced expressionof the humanized CD19 CAR constructs.

Example 9

As discussed above, in some embodiments, CAR constructs comprise adetection tag. However, in several embodiments, no tag is used.Experiments were performed in order to evaluate the expression ofnon-flagged humanized CARS. This experiment employed NK19H-NK-2, -5 and-9 as non-limiting examples of non-flagged humanized CARs. FIGS. 31A-31Bshow expression data (percent expression in 31A, mean fluorescence in31B) of the indicated construct by NK cells from three different donors,measured each week for 4 weeks. These data demonstrate that non-flaggedversions of the CD19 CAR constructs as provided for herein expressrelatively similarly to one another, but more robustly thannon-humanized constructs. Each of the non-flagged humanized constructswere expressed on at least about 70-80% of the NK cells.

Example 10

Experiments to evaluate the cytotoxicity of NK cells expressinghumanized, non-flagged CD19 CAR constructs were performed. An in vitrore-challenge assay was performed as described above, using Raji cells(FIGS. 32A-32B) or Nalm6 cells (FIGS. 32C-32D) as the target cell. FIG.32A shows the percent of Raji cells co-cultured with the indicatedtreatment, measured as a percent of the number of cells measured in aRaji-only control group at day 10 of the experiment. As shown, each ofthe CD19 CAR constructs, whether humanized or not, and whether tagged ornot, substantially eliminated Raji cell growth through Day 10. FIG. 32Bshows the final time point, and there remains limited Raji cell growthin each of the experimental groups with CD19 CARs, though the data showa small trend to greater prevention of Raji growth in with theNK19H-NF-2 and NK19H-NF-5 groups. FIGS. 32C and 32D show thecorresponding data using Nalm6 cells. FIG. 32C shows that, similar toRaji cells, each of the CD19 CAR constructs, whether humanized or not,and whether tagged or not, substantially eliminated Nalm6 cell growththrough 10 days. At the final time point, there was Nalm6 cell growthacross all groups, though the data trends to the humanized constructsbeing more effective at preventing growth.

FIGS. 33A-33J relate to the detected cytokine profile for NK cellsexpressing the indicated constructs. As discussed above, the culturemedia from each treatment group was assayed for interferon gamma(33A/33F), GM-CSF (33B/33G), TNF-alpha (33C/33H), Perforin (33D/331),and Granzyme B (33E/33J). FIGS. 33A-33E show data from the Raji cellrechallenge, while 33F-33J show data from the Nalm6 rechallenge. Asshown, the non-flagged, humanized CD19 CAR expressing NK cells releasesimilar levels of similar amount of IFN-gamma into the media, with thoselevels being slightly greater than the amount released by NK cellsexpressing the non-humanized NK19-1 CAR. Similarly, GM-CSF release wasfairly consistent among the humanized, non-flagged CAR bearing NK cells,and at a level slightly about the NK19-1 expressing cells. TNF-alpharelease showed a similar pattern, while perforin release was consistentamong all the CD19 CAR expressing NK cells, and at a level belowcontrol. Lastly in the Raji cells, granzyme B showed a similar degree ofrelease for the non-flagged, humanized CD19 CARs, at a level slightlyabove the NK19-1 expressing cells. For the most part, the data collectedfrom the Nalm6 experiment showed similar patterns. According to severalembodiments, the greater degree of release of cytotoxicity-mediatingcytokines leads to a more effective therapeutic. In some embodiments,the greater degree of cytokine release is due, at least in part, to theenhanced expression of the non-flagged, humanized CD19 CAR constructs bythe NK cells and/or due, at least in part, to the enhanced persistenceof the NK cells expressing the non-flagged, humanized CD19 CARconstructs. Further supporting these concepts, FIG. 34 shows datarelated to the persistence of NK cells expressing the indicatednon-flagged, humanized constructs over four weeks in culture, ascompared to NK cells expressing NK19-1. While the overall cell countsappear similar among the NK cells expressing the humanized constructs,the cell counts show the same basic pattern as the cytokines, that is,slightly greater than NK cells expressing non-humanized NK19-1.

Example 11

Similar to the experiments above, an in vivo assessment of the efficacyof non-flagged, humanized constructs was performed. FIG. 35A shows aschematic of the protocol used. FIG. 35B shows the in vivobioluminescence measurements, which show that humanized, non-flaggedconstructs appear to slow tumor progression to a greater extent than NKcells expressing the NK19-1 construct. FIG. 35C, recapitulates theimaging data in a line graph, where the enhanced inhibition of tumorcell growth can clearly be seen when the mice received NK cellsexpressing any of the non-flagged, humanized CD19 CAR constructs. FIG.35D reflects the enhanced expression of the non-flagged, humanizedconstructs by NK cells. FIGS. 36A-36F relate to the persistence of NKcells expressing the indicated constructs over the timeline of theexperiment. FIG. 36A indicates that NK cells expressing the non-flagged,humanized CD19 CAR constructs are more persistent in vivo, making up alarger percentage of the overall CD56+ cells in the peripheral blood atDay 13. FIG. 36B demonstrates that, at Day 13, NK cells expressing anyCD19 CAR inhibit tumor growth better than control, with the that NKcells expressing the non-flagged, humanized CD19 CAR constructs showinga slight advantage over the NK19-1 expressing NK cells in terms oflimiting CD19+ tumor cell growth. FIG. 36C shows similar data to 36B,using GFP positive tumor cell count as the benchmark. FIG. 36D showspersistence data at day 27, wherein the NK19-NF-2 and -9 expressing NKcells have elevated population numbers compared to the other groups,indicating enhanced persistence over a longer period of time. Similar tothe earlier time-point, FIGS. 36E and 36F show that the CD19-targetingCAR constructs are all effective at limiting tumor cell growth, comparedto controls. According to several embodiments provided for herein,expression of a non-flagged, humanized CD19 targeting CAR by NK cellsengenders those NK cells with enhanced in vivo persistence andcytotoxicity.

Example 11

As discussed herein, in several embodiments engineered NK cells areprepared for allogeneic cell therapy. As such, in several embodiments,the engineered NK cells to be administered are prepared and then frozenfor later use in a subject. Experiments were performed to determinewhether the process of cryopreservation followed by thawing wouldadversely impact the engineered NK cells, such as by reducing theirviability, persistence or cytotoxicity. FIG. 37A shows the schematicexperimental protocol employed, as well as the experimental groups andother conditions used. For cells with an “IL12/IL18” designation, thecells were expanded in the presence of soluble IL12 and/or IL18, asdescribed in in U.S. Provisional Patent Application No. 62/881,311,filed Jul. 31, 2019 and Application No. 62/932,342, filed Nov. 7, 2019,each of which is incorporated in its entirety by reference herein. FIGS.37B and 37C shows the in vivo bioluminescence imaging from the indicatedexperimental groups. FIG. 38A-38H show line graphs that reflect thebioluminescence intensity over time. These data are recapitulated inFIG. 38I, which shows the first 30 days, and FIG. 38J which shows datathrough 56 days. While FIG. 38I shows a clear distinction between the NKcells expressing CD19 CARs and the controls, there is nominal separationamong the experimental groups. However, FIG. 38J shows data through 56days, and there is a greater distinction of the ability of NK cellsexpressing the various CAR constructs and processed under the indicatedconditions at inhibiting tumor cell growth. Of note is that the “pairs”of groups (same CAR construct, fresh vs. frozen) show fairly similaranti-tumor activity. This indicates, that, according to severalembodiments, engineered NK cells expressing anti-CD19 CARs are effectivenot only when prepared and administered fresh. Additionally, accordingto several embodiments, engineered NK cells expressing anti-CD19 CARsare effective not only when prepared, frozen, then thawed andadministered (e.g., as in an allogeneic context).

FIG. 39 shows a line graph of body mass of the mice treated with theindicated constructs over 56 days of the experiment. A reduction in bodyweight is correlated with increased tumor growth, e.g., progression ofthe tumor results in a decreased health of the mice, and correspondingloss of body weight (e.g., wasting). As shown, the control groups showsubstantial loss of body mass by 30 days, while experimental groups areincreasing in body mass for the majority of the experiment. As with thebioluminescence data discussed above, there is a notable trend that manyof the fresh versus frozen preparations exhibit substantially similareffects on body weight. According to several embodiments, engineered NKcells expressing anti-CD19 CARs are effective not only when prepared andadministered fresh. Additionally, according to several embodiments,engineered NK cells expressing anti-CD19 CARs are effective not onlywhen prepared, frozen, then thawed and administered (e.g., as in anallogeneic context).

It is contemplated that various combinations or subcombinations of thespecific features and aspects of the embodiments disclosed above may bemade and still fall within one or more of the inventions. Further, thedisclosure herein of any particular feature, aspect, method, property,characteristic, quality, attribute, element, or the like in connectionwith an embodiment can be used in all other embodiments set forthherein. Accordingly, it should be understood that various features andaspects of the disclosed embodiments can be combined with or substitutedfor one another in order to form varying modes of the disclosedinventions. Thus, it is intended that the scope of the presentinventions herein disclosed should not be limited by the particulardisclosed embodiments described above. Moreover, while the invention issusceptible to various modifications, and alternative forms, specificexamples thereof have been shown in the drawings and are hereindescribed in detail. It should be understood, however, that theinvention is not to be limited to the particular forms or methodsdisclosed, but to the contrary, the invention is to cover allmodifications, equivalents, and alternatives falling within the spiritand scope of the various embodiments described and the appended claims.Any methods disclosed herein need not be performed in the order recited.The methods disclosed herein include certain actions taken by apractitioner; however, they can also include any third-party instructionof those actions, either expressly or by implication. In addition, wherefeatures or aspects of the disclosure are described in terms of Markushgroups, those skilled in the art will recognize that the disclosure isalso thereby described in terms of any individual member or subgroup ofmembers of the Markush group.

The ranges disclosed herein also encompass any and all overlap,sub-ranges, and combinations thereof. Language such as “up to,” “atleast,” “greater than,” “less than,” “between,” and the like includesthe number recited. Numbers preceded by a term such as “about” or“approximately” include the recited numbers. For example, “about 90%”includes “90%.” In some embodiments, at least 95% sequence identity orhomology includes 96%, 97%, 98%, 99%, and 100% sequence identity orhomology to the reference sequence. In addition, when a sequence isdisclosed as “comprising” a nucleotide or amino acid sequence, such areference shall also include, unless otherwise indicated, that thesequence “comprises”, “consists of” or “consists essentially of” therecited sequence.

In several embodiments, there are provided amino acid sequences thatcorrespond to any of the nucleic acids disclosed herein, whileaccounting for degeneracy of the nucleic acid code. Furthermore, thosesequences (whether nucleic acid or amino acid) that vary from thoseexpressly disclosed herein, but have functional similarity orequivalency are also contemplated within the scope of the presentdisclosure. The foregoing includes mutants, truncations, substitutions,or other types of modifications.

Any titles or subheadings used herein are for organization purposes andshould not be used to limit the scope of embodiments disclosed herein.

What is claimed is:
 1. An immune cell that expresses a CD19-directedchimeric antigen receptor (CAR), the chimeric antigen receptorcomprising: an extracellular anti-CD19 binding moiety comprising asingle chain Fragment variable (scFv), wherein the scFv comprises: avariable heavy (VH) domain, and a variable light (VL) domain; a hinge,wherein the hinge is a CD8 alpha hinge; a transmembrane domain, whereinthe transmembrane domain comprises a CD8 alpha transmembrane domain; andan intracellular signaling domain, wherein the intracellular signalingdomain comprises: an OX40 subdomain, wherein the OX40 subdomain isencoded by a sequence having at least 95% sequence identity to SEQ IDNO. 5, a CD3 zeta subdomain, wherein the CD3 zeta subdomain is encodedby a sequence having at least 95% sequence identity to SEQ ID NO. 7, andwherein the polynucleotide also encodes membrane-bound interleukin-15(mbIL15), wherein the mbIL15 is encoded by a sequence having at least95% sequence identity to SEQ ID NO.
 11. 2. The immune cell of claim 1,wherein the immune cell is a Natural Killer (NK) cell, wherein the OX40subdomain is encoded by SEQ ID NO. 5, wherein the CD3 zeta subdomain isencoded by SEQ ID NO. 7, and wherein the mbIL15 is encoded by SEQ ID NO.11.
 3. The immune cell of claim 1, wherein the encoded VH domaincomprises the amino acid sequence of SEQ ID NO:
 120. 4. The immune cellof claim 1, wherein the encoded VL domain comprises the amino acidsequence of SEQ ID NO:
 118. 5. The immune cell of claim 1, wherein theencoded OX40 subdomain comprises the amino acid sequence of SEQ ID NO:6.
 6. The immune cell of claim 1, wherein the encoded CD3 zeta subdomaincomprises the amino acid sequence of SEQ ID NO:
 8. 7. The immune cell ofclaim 1, wherein the CD19-directed CAR has at least 95% sequenceidentity to the amino acid sequence set forth in SEQ ID NO:
 187. 8. Theimmune cell of claim 1, wherein the CD19-directed CAR comprises theamino acid sequence set forth in SEQ ID NO:
 187. 9. The immune cell ofclaim 1, wherein the VL domain comprises a light chain complementaritydetermining region 1 (LC CDR1) of SEQ ID NO: 124, 127, or 130, a lightchain complementarity determining region. 2 (LC CDR2) of SEQ ID NO: 125,128, or 131, and a light chain complementarity determining region 3 (LCCDR3) of SEQ ID NO: 126, 129, or 132, and the VH domain comprises aheavy chain complementarity determining region 1 (HC CDR1) of SEQ ID NO:133, 136, 139, or 142; a heavy chain complementarity determining region2 (HC CDR2) of SEQ ID NO: 134, 137, 140, or 143, and a heavy chaincomplementarity determining region 3 (HC CDR3) of SEQ ID NO: 135, 138,141, or
 144. 10. An immune cell that expresses a CD19-directed chimericantigen receptor, the chimeric antigen receptor comprising: anextracellular anti-CD19 binding moiety, wherein the anti-CD19 bindingmoiety comprises a heavy chain variable (VH) domain and a and a lightchain variable (VL) domain, the VH domain comprising a VH domainresulting from humanization of the VH domain amino acid sequence setforth in SEQ ID NO: 33, the VL domain resulting from humanization of theVL domain amino acid sequence set forth in SEQ ID NO: 32, a hinge and/ortransmembrane domain, and an intracellular signaling domain, wherein theintracellular signaling domain comprises an OX40 subdomain, and whereinthe cell also expresses membrane-bound interleukin-15 (mbIL15).
 11. Theimmune cell of claim 10, wherein the intracellular signaling domainfurther comprises a CD3zeta subdomain.
 12. The immune cell of claim 10,wherein the OX40 subdomain comprises the amino acid sequence of SEQ IDNO: 6 and the CD3zeta subdomain comprises the amino acid sequence of SEQID NO: 8, wherein the VH domain has at least about 80% sequence identityto SEQ ID NO: 33, and wherein the VL domain has at least about 80%sequence identity to SEQ ID NO: 32
 13. The immune cell of claim 10,wherein the immune cell is a Natural Killer (NK) cell, wherein the hingedomain comprises a CD8a hinge domain, wherein the encoded CD8a hingedomain comprises the amino acid sequence of SEQ ID NO: 2, and whereinthe encoded mbIL15 comprises the amino acid sequence of SEQ ID NO: 12.14. The immune cell of claim 10, wherein the immune cell is a NK cell,and wherein the VL domain comprises a light chain complementaritydetermining region 1 (LC CDR1) of SEQ ID NO: 124, 127, or 130, a lightchain complementarity determining region. 2 (LC CDR2) of SEQ ID NO: 125,128, or 131, and a light chain complementarity determining region 3 (LCCDR3) of SEQ ID NO: 126, 129, or 132, and wherein the VH domaincomprises a heavy chain complementarity determining region 1 (HC CDR1)of SEQ ID NO: 133, 136, 139, or 142; a heavy chain complementaritydetermining region 2 (HC CDR2) of SEQ ID NO: 134, 137, 140, or 143, anda heavy chain complementarity determining region 3 (HC CDR3) of SEQ IDNO: 135, 138, 141, or
 144. 15. An immune cell that expresses aCD19-directed chimeric antigen receptor, the chimeric antigen receptorcomprising: an extracellular anti-CD19 binding moiety comprising asingle chain Fragment variable (scFv), wherein the scFv comprises: avariable heavy (VH) domain, wherein the encoded VH domain comprises anamino acid sequence having at least 95% sequence identity to SEQ ID NO:120; and a variable light (VL) domain, wherein the encoded VL domaincomprises an amino acid sequence having at least 95% sequence identityto SEQ ID NO: 118; a hinge; a transmembrane domain; and an intracellularsignaling domain, wherein the intracellular signaling domain comprises:an OX40 subdomain, a CD3 zeta subdomain; and wherein the polynucleotidealso encodes membrane-bound interleukin-15 (mbIL15), wherein the mbIL15is encoded by a sequence having at least 95% sequence identity to SEQ IDNO.
 11. 16. The immune cell of claim 15, wherein the OX40 subdomain isencoded by a sequence having at least 95% sequence identity to SEQ IDNO.
 5. 17. The immune cell of claim 15, wherein the CD3 zeta subdomainis encoded by a sequence having at least 95% sequence identity to SEQ IDNO.
 7. 18. The immune cell of claim 15, wherein the VL domain comprisesa light chain complementarity determining region 1 (LC CDR1) of SEQ IDNO: 124, 127, or 130, a light chain complementarity determining region.2 (LC CDR2) of SEQ ID NO: 125, 128, or 131, and a light chaincomplementarity determining region 3 (LC CDR3) of SEQ ID NO: 126, 129,or 132, and wherein the VH domain comprises a heavy chaincomplementarity determining region 1 (HC CDR1) of SEQ ID NO: 133, 136,139, or 142; a heavy chain complementarity determining region 2 (HCCDR2) of SEQ ID NO: 134, 137, 140, or 143, and a heavy chaincomplementarity determining region 3 (HC CDR3) of SEQ ID NO: 135, 138,141, or
 144. 19. The immune cell of claim 15, wherein the encoded VHdomain comprises the amino acid sequence of SEQ ID NO:
 120. 20. Theimmune cell of claim 15, wherein the encoded VL domain comprises theamino acid sequence of SEQ ID NO: 118.